U.S. patent application number 12/583579 was filed with the patent office on 2010-04-01 for oncogenic-ras-signal dependent lethal compounds.
Invention is credited to Rohitha SriRamaratnam, Brent R. Stockwell, Wan Seok Yang.
Application Number | 20100081654 12/583579 |
Document ID | / |
Family ID | 39710676 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100081654 |
Kind Code |
A1 |
Stockwell; Brent R. ; et
al. |
April 1, 2010 |
Oncogenic-RAS-signal dependent lethal compounds
Abstract
Compounds with cancer cell specific lethality are provided. In
particular, RAS-selective lethal compounds and compositions are
provided. Also provided are methods of screening for such compounds
and methods of treating a condition in a mammal, by administering
to the mammal a therapeutically effective amount of such compounds
or compositions.
Inventors: |
Stockwell; Brent R.; (New
York, NY) ; Yang; Wan Seok; (New York, NY) ;
SriRamaratnam; Rohitha; (New York, NY) |
Correspondence
Address: |
BRYAN CAVE LLP
1290 AVENUE OF THE AMERICAS
NEW YORK
NY
10104
US
|
Family ID: |
39710676 |
Appl. No.: |
12/583579 |
Filed: |
August 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US08/02390 |
Feb 21, 2008 |
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12583579 |
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60902575 |
Feb 21, 2007 |
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Current U.S.
Class: |
514/229.5 ;
514/292; 544/99; 546/85 |
Current CPC
Class: |
C07D 471/04
20130101 |
Class at
Publication: |
514/229.5 ;
546/85; 514/292; 544/99 |
International
Class: |
A61K 31/5365 20060101
A61K031/5365; C07D 471/04 20060101 C07D471/04; A61K 31/437 20060101
A61K031/437; C07D 498/14 20060101 C07D498/14 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under grant
no. 1 R01 CA097061-01 awarded by the National Institutes of
Health/National Cancer Institute. The government has certain rights
in the invention.
Claims
1. A compound having the formula X: ##STR00113## wherein R.sub.1,
R.sub.2, R.sub.3, and R.sub.6 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl, wherein each alkyl, alkoxy, aralkyl, carbocyclic,
heterocyclic, aryl, heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl is optionally substituted with at least one
substituent; R.sub.4 and R.sub.5 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle, wherein each
alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, and alkylheterocycle may be optionally substituted
with at least one substituent; R.sup.8 is selected from H,
C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
2. The compound according to claim 1 wherein R.sub.2 is a
substituted aryl.
3. The compound according to claim 1 wherein R.sub.2 is a
halo-substituted aryl.
4. The compound according to claim 1 wherein R.sub.2 is a
tetra-substituted aryl.
5. The compound according to claim 1, wherein R.sub.2 is a
tetra-fluoro substituted aryl.
6. The compound according to claim 1, wherein R.sub.2 is a
C.sub.1-8 alkyl substituted with carbonyl.
7. The compound according to claim 1, wherein each optional
substituent is independently selected from the group consisting of
H, cyano, oxo, nitro, acyl, acylamino, halogen, hydroxy, amino
acid, amine, amide, carbamate, ester, ether, carboxilic acid, thio,
thioalkyl, thioester, thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy,
C.sub.1-8alkenyl, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic,
3- to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, and arylsulfonyl.
8. A compound according to claim 1 having the formula: ##STR00114##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
9. A compound selected from the group consisting of: ##STR00115##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
10. The compound according to claim 9, which is: ##STR00116## or a
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof.
11. The compound according to claim 10, which is enantiomerically
pure.
12-15. (canceled)
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an enantiomerically pure RAS-selective
lethal compound having the structure: ##STR00117## or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
17. A composition comprising a pharmaceutically acceptable carrier
and a RAS-selective lethal compound according to formula XI:
##STR00118## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
and R.sub.6 are independently selected from H, Halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
18-23. (canceled)
24. A compound having the formula XII: ##STR00119## wherein R.sub.2
and R.sub.3 are independently selected from H, methyl, methyl
benzoate, propargyl, and phenyl, wherein at least one of R.sub.2
and R.sub.3 is other than H; R.sub.6, R.sub.8, and R.sub.9 are
independently selected from H, halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached; wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
25-27. (canceled)
28. A compound having the formula XIII: ##STR00120## wherein
R.sub.2 is selected from halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3-
to 8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, carboxylate,
ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether, wherein each
alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
29-34. (canceled)
35. A compound having the formula XIV: ##STR00121## wherein R.sub.2
and R.sub.3 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether and is
optionally substituted with at least one substituent, and wherein
at least one of R.sub.2 and R.sub.3 is other than H; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; n is 0 or 1; E is
an electrophilic group that has a lower electrophilicity index
value than does Cl; wherein R.sub.2 and R.sub.3 are not the same
group, or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
36-43. (canceled)
44. A compound according to formula XV: ##STR00122## wherein
R.sub.6, R.sub.8, and R.sub.9 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; and R.sub.10 is
selected from the group consisting of a C.sub.1-8 ester and an
acid, which ester or acid is hydrolyzable in vivo; or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
45. A compound according to claim 44, wherein each substituent for
is independently selected from the group consisting of cyano, oxo,
nitro, acyl, acylamino, halogen, hydroxy, amino acid, amine, amide,
carbamate, ester, ether, carboxylic acid, thio, thioalkyl,
thioester, thioether, C.sub.1-8alkyl, C.sub.1-8alkene,
C.sub.1-8alkyne, C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to
8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, sulfate,
sulfonamide, sulfoxide, sulfonate, sulfone, alkylsulfonyl,
arylsulfonyl, C.sub.1-8alkoxy alkyne, C.sub.1-8 alkyl alkyne, and
C.sub.1-8 alkyne acetate.
46. A compound according to claim 45, wherein R.sub.6, R.sub.8, and
R.sub.9 are each H.
47. A compound according to claim 45, wherein R.sub.10 is --COOH or
--C(.dbd.O)OCH.sub.3.
48. A compound selected from the group consisting of: ##STR00123##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
49. The compound according to claim 48, which is: ##STR00124## or a
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof.
50. The compound according to claim 49, which is enantiomerically
pure.
51-52. (canceled)
53. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an enantiomerically pure RAS-selective
lethal compound having the structure: ##STR00125## or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
54. A composition comprising a RAS-selective lethal compound
according to claim 1, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
55-56. (canceled)
57. A method of treating a condition in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
compound according to claim 1, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
58-92. (canceled)
93. A method for identifying RAS-selectively lethal compounds
comprising: (a) determining a % GI of candidate compounds in a BJ
cell system comprising a plurality of isogenic cell lines at least
one of which is non-tumorigenic and at least one of which is
tumorigenic, wherein a % GI of at least 90% at a concentration of
about 10 .mu.M or less in at least one tumorigenic BJ cell line is
considered to be a lethal compound, (b) determining a potency
profile, in the BJ cell system, of the candidate compounds from (a)
determined to be lethal, wherein those candidate compounds having
at least a 4-fold differential potency between non-tumorigenic and
tumorigenic cell lines in the BJ cell system are considered to be
selective, and (c) identifying the selective candidate compounds
from (b), which have a similar potency profile for each of the
tumorigenic cell lines in the BJ cell system, wherein those
candidate compounds having a similar potency profile are considered
to be RAS-selectively lethal compounds.
94-118. (canceled)
119. A composition comprising a RAS-selective lethal compound
according to claim 44, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
120. A method of treating a condition in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
compound according to claim 44, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of International
Application No. PCT/US2008/002390, which was filed on Feb. 21,
2008, which claims the benefit of U.S. Provisional Application No.
60/902,575, which was filed on Feb. 21, 2007, both of which are
incorporated herein in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to compounds with cancer cell
specific lethality. In particular, the present invention relates to
RAS-selective lethal compounds and compositions. The present
invention also relates to methods of screening for such compounds
and methods of treating a condition in a mammal, e.g., by
administering to the mammal a therapeutically effective amount of
such compounds or compositions.
BACKGROUND OF THE INVENTION
[0004] Cancer can be effectively treated using targeted therapy.
There are increasing efforts to fulfill the promise of targeted
therapy using antibodies, peptides, and small molecules that
selectively affect cancer cells. The key to successful targeted
therapy is to identify target molecules that play a unique role in
tumor cells. Tumor cells have a broad spectrum of mutations and
chromosomal rearrangements affecting gene products that play
critical roles in the genesis and maintenance of tumors (Hanahan
and Weinberg, 2000; Vogelstein and Kinzler, 2004). Some of these
mutated gene products are shared among cancer cells from different
origins and may be good targets for developing anti-cancer drugs
with a high therapeutic index.
[0005] Leads for new cancer therapeutics often emerge from
target-based in vitro screening. For example, compounds that
interfere with post-translational modifications of RAS, such as
Farensyl Transferase Inhibitors (FTIs) are in clinical trials. In
addition, targeted kinase inhibitors, such as Gleevec (Capdeville
et al., 2002) and Sorafenib (Wilhelm et al., 2006) have been
effective in treating tumors using a targeted therapy approach.
[0006] Many proteins cannot be targeted with enzymatic, active-site
inhibitors. However, in some cases, the oncogenic functions of
these targets can be inhibited by small molecules that disrupt
their interaction with other necessary protein partners or other
aspects of the protein's function. Small molecules that disrupt the
interaction between the tumor suppressor p53 and the oncoprotein
MDM2, or between pro-apoptotic and anti-apoptotic bcl-2 family
proteins, induce selective death in a subset of tumor cells (Fry
and Vassilev, 2005). In a more challenging approach to restore a
mutant protein's function, others have reported the identification
of pharmacological chaperones that convert the conformation of
mutant p53 into wild type (Foster et al., 1999).
[0007] A complementary approach to such target-based discovery
strategies is synthetic lethal screening. Activation of multiple
downstream signaling molecules is crucial to RAS-mediated
tumorigenesis and tumor maintenance. Synthetic lethal screening is
a strategy for revealing critical RAS-linked targets. (Hartwell et
al., 1997). For a given mutation "A", if there exists a second
mutation "B" that is particularly lethal to the organism in the
presence of A, mutation B is synthetic lethal with mutation A
because the lethality requires the synthesis, or bringing together,
of the two mutations. In synthetic lethal screening with oncogenic
RAS, the second perturbation can be created by using a small
molecule to alter the function of a target protein, rather than by
introducing a mutation in the gene encoding the protein.
(Stockwell, 2000). The existence of striking synthetic lethal
interactions in genome-wide studies has been demonstrated in the
work of Tong et al. with yeast deletion strains (Tong et al.,
2001).
[0008] Classic yeast synthetic lethal screening involves cDNA
library transformation, genome wide mutagenesis and colony-color
assays in order to identify clones that are necessary for the
survival of yeast cells with a given mutation (Barbour and Xiao,
2006); alternatively, use of gene deletion panels has been
successful (Tong et al., 2001). Although these classic methods have
been useful in elucidating functional genetic connectivity in model
organisms, they are not as applicable to mammalian systems. Small
molecules, and additionally RNAi libraries using siRNAs or shRNAs
(Moffat and Sabatini, 2006), are more tractable than mutagenesis
for carrying out synthetic lethal screening in cell culture
systems.
[0009] Unlike synthetic lethal screening with mutations, synthetic
lethal screening with small organic molecules does not provide
direct information about the nature of the two mutations that cause
synthetic lethality. The relevant target of each compound must
ultimately be identified.
[0010] However, the use of small molecules in place of gene
deletions offers a number of advantages that can overcome the lack
of direct information about the target protein. First, compound
addition can be varied in time and concentration, providing finer
control of the perturbation to the target cell. Second, small
molecules can induce a gain-of-function or affect just one domain
of a protein, unlike deletion mutations or RNA interference (RNAi).
Third, it is more straightforward to develop a chemotherapeutic
reagent from a small molecule lead than from a gene deletion or
RNAi reagent. For these reasons, the concept of synthetic lethal
screening with small molecules is complementary to such studies
with gene deletions or RNAi. Indeed, several groups have reported
the discovery of small-molecules that are synthetic lethal to
mutations such as p53 (Stockwell et al., 1999), ERBB2 (Fantin et
al., 2002), p21 (Torrance et al., 2001), and DPC4 (Wang et al.,
2006). Further optimization of such hit compounds may yield
anti-cancer leads with high therapeutic indices.
[0011] Synthetic lethal screening using RAS oncogenes has been
studied (Dolma et al., 2003). Considering its critical role in
cancer development, mutant RAS has been the focus of much research
(Barbacid, 1987; Malumbres and Barbacid, 2003; Shaw and Cantley,
2006). Because restoring GTPase activity to mutant RAS is a
challenging task for small molecules, efforts have focused on the
more feasible approach of inhibiting post-translational processing
of the RAS proteins to inactivate their oncogenic signaling. The
C-terminal four amino acids of RAS proteins, i.e. the CAAX motif,
are conjugated to a fifteen carbon isoprenoid by
farensyltransferase (FTase) in the endoplasmic reticulum. This
allows RAS proteins to be anchored in the plasma membrane, which is
essential for activity (Schafer et al., 1989; Schafer et al.,
1990). Several `CAAX peptide mimetic` compounds have been developed
as FTIs and have shown promising results in pre-clinical mouse
studies (Kohl et al., 1995; Nagasu et al., 1995). However, these
compounds showed mixed results in clinical trials, possibly because
KRAS and NRAS, whose mutations are more prevalent than HRAS in
human cancer, can be conjugated to a twenty carbon isoprenoid
modification by geranylgeranyltransferase upon inhibition of FTase
activity (Rowell et al., 1997; Whyte et al., 1997).
[0012] An alternative approach using `S-farensyl cystein mimetic`
compounds, such as FTS, has been reported to work on all three RAS
proteins and to be less cytotoxic than FTIs (Marom et al., 1995).
These compounds were identified as inhibitors of isoprenylcysteine
carboxylmethyltransferase (ICMT), another enzyme involved in RAS
protein maturation. However, these compounds are reported to have
the `off-target` effect of dislodging membrane-bound RAS proteins,
which leads to inhibition of RAS signal activation (Marciano et
al., 1995; Marom et al., 1995). The anti-tumor effect of FTIs and
FTSs is significant in pre-clinical experiments and research is
on-going to improve the efficacy of these reagents in clinical
settings. However, there has been debate as to whether inhibition
of RAS post-translational processing will result in cancer-cell
selective inhibition, given that other proteins are also subject to
the same lipid modifications.
[0013] The compound erastin was found to display synthetic
lethality with oncogenic RAS. As expected, the target of erastin is
not RAS itself: erastin binds to voltage-dependant anion channels,
a novel target for anti-cancer drugs to induce
RAS-RAF-MEK-dependent oxidative, non-apoptotic cell death. (Yagoda
et al., 2007).
[0014] Accordingly, there exists a significant need to screen,
identify, and/or develop compounds and compositions that
selectively target tumor cells, especially RAS-selective lethal
compounds and compositions.
SUMMARY OF THE INVENTION
[0015] To circumvent concerns with the target-based approach in the
RAS field, the complementary approach of synthetic lethal screening
with small molecules was used to identify those compounds that
display synthetic lethality with oncogenic RAS. Novel
small-molecules that have synthetic lethal interactions with mutant
RAS have been identified and characterized. Moreover, their
mechanisms of action have also been characterized.
[0016] Accordingly, one embodiment of the present invention is a
compound having the formula I:
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.6 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, C.sub.1-8aralkyl,
3- to 8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, acyl,
alkylsulfonyl, and arylsulfonyl, wherein each alkyl, alkoxy,
aralkyl, carbocyclic, heterocyclic, aryl, heteroaryl, acyl,
alkylsulfonyl, and arylsulfonyl is optionally substituted with at
least one substituent; R.sub.4 and R.sub.5 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0017] An additional embodiment of the present invention is a
compound of the formula:
##STR00002##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0018] Another embodiment of the present invention is a compound of
the formula:
##STR00003##
or an enantiomer, optical isomer; diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0019] Another embodiment of the present invention is a
pharmaceutical composition that includes a pharmaceutically
acceptable carrier and one or more compounds of the present
invention, including for example compounds of formula I, Ia, and
Ib, as well as, compounds 1 and 2.
[0020] A further embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound of formula II:
##STR00004##
wherein R.sub.1 and R.sub.2 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.3R.sup.4, OC(R.sup.3).sub.2COOH, SC(R.sup.3).sub.2COOH,
NHCHR.sup.3COOH, COR.sup.4, CO.sub.2R.sup.4, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.3R.sup.4,
OC(R.sup.3).sub.2COOH, SC(R.sup.3).sub.2COOH, NHCHR.sup.3COOH,
COR.sup.4, CO.sub.2R.sup.4, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.3 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.4 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkynyl, aryl, carbocycle, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle, and
heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic may be optionally substituted
with at least one substituent; X is selected from halo and
C.sub.1-8 alkyl; and n is 0-8, or an enantiomer, optical isomer,
diastereomer, N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0021] An additional embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound of formula III:
##STR00005##
wherein R.sub.1 is selected from H, halo, C.sub.1-8 alkyl,
C.sub.1-8alkylene, 1-hydroxyl-.beta.-D-glucose optionally
substituted with from 1-4 acetates and/or
1-hydroxyl-.beta.-D-glucose tetraacetate, and
1-thionyl-.beta.-D-glucose optionally substituted with from 1-4
acetates, 0, and/or C.sub.1-8 alkoxy; R.sub.2 is selected from H,
halo, C.sub.1 alkyl, and 1-thionyl-.beta.-D-glucose substituted
with from 1-4 acetates, O, or C.sub.1-8 alkoxy; R.sub.3 and R.sub.4
are independently selected from H, halo, C.sub.1-8 alkyl, C.sub.1-8
alkoxy, 1-hydroxyl-.beta.-D-glucose optionally substituted with
from 1-4 acetates and/or 1-hydroxyl-.beta.-D-glucose tetraacetate,
and 1-thionyl-.beta.-D-glucose optionally substituted with from 1-4
acetates; R.sub.5 and R.sub.6 are independently selected from H,
OH, and acetate esters; and R.sub.7 is selected from H or carbonyl,
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0022] Another embodiment of the present invention is a composition
containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula IV:
##STR00006##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
are independently selected from H, Halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0023] A further embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula V:
##STR00007##
wherein R.sub.1 is independently selected from H, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.2R.sup.3,
OC(R.sup.2).sub.2COOH, SC(R.sup.2).sub.2COOH, NHCHR.sup.2COOH,
COR.sup.3, CO.sub.2R.sup.3, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.2R.sup.3,
OC(R.sup.2).sub.2COOH, SC(R.sup.2).sub.2COOH, NHCHR.sup.2COOH,
COR.sup.3, CO.sub.2R.sup.3, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.2 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.3 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and
A is C, N, or S, or
[0024] an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0025] An embodiment of the present invention is a compound having
the formula VI:
wherein
##STR00008##
R.sub.2 and R.sub.3 are independently selected from H, methyl,
methyl benzoate, propargyl, and phenyl, wherein at least one of
R.sub.2 and R.sub.3 is other than H; R.sub.6, R.sub.8, and R.sub.9
are independently selected from H, halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached; or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0026] In another embodiment of the present invention, a compound
is provided having the formula VII:
##STR00009##
wherein R.sub.2 is selected from halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether, wherein each
alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0027] A further embodiment of the present invention is a compound
having the formula VIII:
##STR00010##
wherein R.sub.2 and R.sub.3 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether and is
optionally substituted with at least one substituent, and wherein
at least one of R.sub.2 and R.sub.3 is other than H; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; n is 0 or 1; E is
an electrophilic group that has a lower electrophilicity index
value than does Cl; or an enantiomer, optical isomer, diastereomer,
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof.
[0028] In a further embodiment of the present invention is a
compound according to formula IX:
##STR00011##
wherein R.sub.6, R.sub.8, and R.sub.9 are independently selected
from H, halo, C.sub.1-10alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; and R.sub.10 is
selected from the group consisting of a C.sub.1-8 ester and an
acid, which ester or acid is hydrolyzable in vivo; or an
enantiomer, optical isomer, diastereomer, N-oxide, crystalline
form, hydrate, or pharmaceutically acceptable salt thereof.
[0029] Compositions are provided comprising a pharmaceutical
carrier and a compound selected from the group consisting of
compounds of formula VI, compounds of formula VII, compounds of
formula VIII, and compounds of formula IX.
[0030] Methods of treating a condition in a mammal are provided,
comprising administering to the mammal a therapeutically effective
amount of a compound selected from the group consisting of
compounds of formula VI, compounds of formula VII, compounds of
formula VIII, and compounds of formula IX.
[0031] Surprisingly, it has been found that only specific
stereoisomers of certain of the compounds of the present invention
are biologically active, e.g., exhibit selective lethality toward
tumor cells. Accordingly, as set forth in more detail below, such
stereoisomers are also part of the present invention.
[0032] For example, another embodiment of the present invention is
a compound having the formula X:
##STR00012##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.6 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, C.sub.1-8aralkyl,
3- to 8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, acyl,
alkylsulfonyl, and arylsulfonyl, wherein each alkyl, alkoxy,
aralkyl, carbocyclic, heterocyclic, aryl, heteroaryl, acyl,
alkylsulfonyl, and arylsulfonyl is optionally substituted with at
least one substituent; R.sub.4 and R.sub.5 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
[0033] In a further embodiment of the invention, a compound is
selected from the group consisting of:
##STR00013##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0034] A preferable embodiment of the present invention is an
enantiomerically pure compound having the structure:
##STR00014##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0035] One preferred compound of formula X is:
##STR00015##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0036] Another preferred compound of formula X is:
##STR00016##
wherein the phenyl ring is optionally substituted with
methoxycarbonyl, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0037] Another embodiment of the present invention is a composition
comprising a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula XI:
##STR00017##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
are independently selected from H, Halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
[0038] Another embodiment of the present invention is a compound
having the formula XII:
##STR00018##
wherein R.sub.2 and R.sub.3 are independently selected from H,
methyl, methyl benzoate, propargyl, and phenyl, wherein at least
one of R.sub.2 and R.sub.3 is other than H; R.sub.6, R.sub.8, and
R.sub.9 are independently selected from H, halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached; wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
[0039] In another embodiment of the present invention, a compound
is provided having the formula XIII:
##STR00019##
wherein R.sub.2 is selected from halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether, wherein each
alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0040] A further embodiment of the present invention is a compound
having the formula XIV:
##STR00020##
wherein R.sub.2 and R.sub.3 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R').sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether and is
optionally substituted with at least one substituent, and wherein
at least one of R.sub.2 and R.sub.3 is other than H; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; n is 0 or 1; E is
an electrophilic group that has a lower electrophilicity index
value than does Cl; wherein R.sub.2 and R.sub.3 are not the same
group, or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0041] A further embodiment of the present invention is a compound
according to formula XV:
wherein
##STR00021##
R.sub.6, R.sub.8, and R.sub.9 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; and R.sub.10 is
selected from the group consisting of a C.sub.1-8 ester and an
acid, which ester or acid is hydrolyzable in vivo; or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0042] In another aspect of the present invention, a compound is
provided which is selected from the group consisting of:
##STR00022##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0043] A further preferred embodiment is an enantiomerically pure
compound having the structure:
##STR00023##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0044] A preferred embodiment of the present invention is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an enantiomerically pure RAS-selective lethal compound
having the structure:
##STR00024##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0045] In one aspect of this embodiment, the compound according to
formula XV has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8alkyl, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne,
C.sub.1-8 alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0046] Other embodiments of the present invention include methods
of treating a condition in a mammal. These methods include the step
of administering to the mammal a therapeutically effective amount
of a compound or composition as described above. In one preferred
embodiment, the condition is cancer. In another preferred
embodiment, the condition is characterized by cells with enhanced
RAS signaling activity.
[0047] An additional embodiment of the present invention is a
method for identifying RAS-selectively lethal compounds. This
method includes the steps of:
[0048] (a) determining a % GI of candidate compounds in a BJ cell
system comprising a plurality of isogenic cell lines at least one
of which is non-tumorigenic and at least one of which is
tumorigenic, wherein a % GI of at least 90% at a concentration of
about 10 .mu.M or less in at least one tumorigenic BJ cell line is
considered to be a lethal compound,
[0049] (b) determining a potency profile, in the BJ cell system, of
the candidate compounds from (a) determined to be lethal, wherein
those candidate compounds having at least a 4-fold differential
potency between non-tumorigenic and tumorigenic cell lines in the
BJ cell system are considered to be selective, and
[0050] (c) identifying the selective candidate compounds from (b),
which have a similar potency profile for each of the tumorigenic
cell lines in the BJ cell system, wherein those candidate compounds
having a similar potency profile are considered to be
RAS-selectively lethal compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] The file of this patent or patent application contains at
least one drawing executed in color. Copies of this patent or
patent application with color drawing(s) will be provided by the
United States Patent and Trademark Office upon request and payment
of the necessary fee.
[0052] FIG. 1 shows seven compounds that were equally active in
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells and
BJ-TERT/LT/ST/RAS.sup.V12 cells in an Alamar blue assay DRD counter
screen. These compounds are divided into 4 groups based on their
structures.
[0053] FIG. 2A shows the selectivity and potency of compounds of
Formula II and other structurally related analogs (including
Compounds 3, 4, 5, and 47324-12) using an Alamar blue assay in
BJ-TERT (eH), BJ-TERT/LT/ST (eHLT), BJ-TERT/LT/ST/RAS.sup.V12
(eLR), and DRD cells.
[0054] FIG. 2B shows the selectivity and potency of Compound 3
using an Alamar blue assay in TRE, TRE/E6/E7/ST, and
TRE/E6/E7/ST/RAS.sup.v12 cells (left panel) and in A549, Calu-1,
PaCa, and HCT116 cells (right panel).
[0055] FIG. 3 shows the selectivity and potency of Compound 3 using
a ViCell (Trypan blue exclusion) assay.
[0056] FIG. 4 shows the selectivity and potency of additional
Formula II compounds and/or other structurally related analogs
using an Alamar blue assay in the BJ-TERT (eH), BJ-TERT/LT/ST
(eHLT), BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0057] FIG. 5 shows the results of counter screening Compound 3
with 36 biologically active compounds, including antioxidants, iron
chelators, and protein synthesis inhibitors in
BJ-TERT/LT/ST/RAS.sup.V12 cells.
[0058] FIG. 6A shows the selectivity and potency of Compounds of
Formula III (including Compounds 6, 17, and 7) using an Alamar blue
assay in BJ-TERT (eH), BJ-TERT/LT/ST (eHLT),
BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0059] FIG. 6B shows the selectivity and potency of Compound 6
using an Alamar blue assay in TRE, TRE/E6/E7/ST, and
TRE/E6/E7/ST/RAS.sup.v12 cells (left) and in A549, Calu-1, PaCa,
and HCT116 cells (right).
[0060] FIG. 7 shows the selectivity and potency of Compound 6 using
a ViCell (Trypan blue exclusion) assay.
[0061] FIG. 8 shows the results of counter screening Compound 6
with 36 biologically active compounds, including antioxidants, iron
chelators, and protein synthesis inhibitors in
BJ-TERT/LT/ST/RAS.sup.V12 cells.
[0062] FIG. 9 shows the selectivity and potency of additional
Formula III compounds and other structurally related analogs using
an Alamar blue assay in the BJ-TERT (eH), BJ-TERT/LT/ST (eHLT),
BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0063] FIG. 10A shows the selectivity and potency of compounds of
Formula V (including Compounds 36 and 37, as well as 24578-7 and
24578-3) using an Alamar blue assay in BJ-TERT (eH), BJ-TERT/LT/ST
(eHLT), BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0064] FIG. 10B shows the selectivity and potency of Compound 36
using an Alamar blue assay in TRE, TRE/E6/E7/ST, and
TRE/E6/E7/ST/RAS.sup.v12 cells (left panel), in MS1 and SVR cells
(center panel), and in A549, Calu-1, PaCa, and HCT116 cells (right
panel).
[0065] FIG. 10C shows the inability of cell cycle blockers
(apigenin, monastrol, olomoucine, nocodazole, hydroxyurea,
colchicine, and rapamycin) to suppress Compound 36-induced cell
death in BJ-TERT/LT/ST/RAS.sup.V12 cells using an Alamar blue
assay.
[0066] FIG. 11 shows the selectivity and potency of Compound 36
using a ViCell (Trypan blue exclusion) assay.
[0067] FIG. 12 shows the results of counter screening Compound 36
with 36 biologically active compounds, including antioxidants, iron
chelators, and protein synthesis inhibitors in
BJ-TERT/LT/ST/RAS.sup.V12 cells.
[0068] FIG. 13 shows the selectivity and potency of additional
Formula V analogs and other structurally related compounds using an
Alamar blue assay in the BJ-TERT (eH), BJ-TERT/LT/ST (eHLT),
BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0069] FIG. 14A shows the selectivity and potency of compounds of
Formula IV (including Compounds 27, 28, and 19738-13) and other
structurally related compounds using an Alamar blue assay in
BJ-TERT (eH), BJ-TERT/LT/ST (eHLT), BJ-TERT/LT/ST/RAS.sup.V12
(eLR), and BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD)
cells.
[0070] FIG. 14B shows the selectivity and potency of Compound 27
using an Alamar blue assay in TRE, TRE/E6/E7/ST, and
TRE/E6/E7/ST/RAS.sup.v12 cells (left panel) and in A549, Calu-1,
PaCa, and HCT116 cells (right panel).
[0071] FIG. 14C shows the results of NCI60 screening of Compound
27. 18 of the 60 human cell lines tested are shown. These 18 cell
lines include a representative sensitive group and a resistant
group.
[0072] FIG. 14D shows the time-dependent effect of Erastin,
Compound 36, Compound 27, Compound 6, and Compound 3 on cell
viability using a ViCell (Trypan blue exclusion) assay.
[0073] FIG. 14E shows cell growth inhibition of Compound 27 using
an Alamar blue assay in Calu-1, Calu-1pLKO, and Calu-1-509
cells.
[0074] FIG. 15 shows the selectivity and potency of Compound 27
using a ViCell (Trypan blue exclusion) assay.
[0075] FIG. 16 shows the results of NCI60 screening of Compound
27.
[0076] FIG. 17 shows the results of counter screening Compound 27
with 36 biologically active compounds, including antioxidants, iron
chelators, and protein synthesis inhibitors in
BJ-TERT/LT/ST/RAS.sup.V12 cells.
[0077] FIG. 18 shows the selectivity and potency of additional
compounds of Formula V and other structurally related compounds
using an Alamar blue assay in BJ-TERT (eH), BJ-TERT/LT/ST (eHLT),
BJ-TERT/LT/ST/RAS.sup.V12 (eLR), and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells.
[0078] FIG. 19 shows the results of counter screening of Erastin,
Compound 3, Compound 6, Compound 27, and Compound 36 with known
iron chelators in BJ-TERT/LT/ST/RAS.sup.V12 cells.
[0079] FIG. 20A shows the cellular iron levels in BJ-TERT,
BJ-TERT/LT/ST, and BJ-TERT/LT/ST/RAS.sup.V12 cells using the
fluorescent iron sensor, Phen Green SK.
[0080] FIG. 20 B shows the expression level of transferrin receptor
1 (TfR1) in BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS.sup.V12 cells
by Western blot. An antibody against actin was included as a
control for assessing protein loading.
[0081] FIG. 20C shows the cellular iron levels in
BJ-TERT/LT/ST/RAS.sup.V12 cells, treated with indicated compounds
for 24 hours, using the fluorescent iron sensor, Phen Green SK.
[0082] FIG. 20D shows the expression level of transferrin receptor
1 (TfR1) in BJ-TERT/LT/ST/RAS.sup.V12 cells treated with DFOM, 2,
311, Erastin, Compound 36, Compound 6, Compound 27, and Compound 3
by Western blot. An antibody against actin was included as a
control for assessing protein loading.
[0083] FIG. 20E shows a quantitative representation of the
expression level of transferrin receptor 1 (TfR1) in
BJ-TERT/LT/ST/RAS.sup.V12 cells treated with DFOM, 2, 311, Erastin,
Compound 36, Compound 6, Compound 27, and Compound 3 by Western
blot (see FIG. 20D).
[0084] FIG. 20F shows cell growth inhibition in HT1080,
HT1080-vector, and HT1080-TfR1KD cells by Compound 27 (left panel)
or Erastin (right panel) in the presence of 150 .mu.M DFOM, 10
.mu.M U0126, 50 .mu.M SU6656, or 100 .mu.M Vitamin E, as indicated,
using an Alamar blue assay.
[0085] FIG. 20G shows cell growth inhibition of Erastin, Compound
3, Compound 27, and Compound 36 (from left to right) in HT1080,
HT1080-vector, and HT1080-TfR1KD cells using an Alamar blue
assay.
[0086] FIG. 21A shows the expression level of TfR1 in HT1080,
HT1080-vector, and HT1080-TfR1KD cells by Western blot analysis
with an antibody directed against TfR1. An antibody against actin
was included as a control for assessing protein loading.
[0087] FIG. 21B shows the level of the basal iron pool in HT1080,
HT1080-vector, and HT1080-TfR1KD cells.
[0088] FIG. 22 shows the results of NCI60 screening of Erastin.
[0089] FIG. 23 shows the results of NCI60 screening of Compound
36.
[0090] FIG. 24 shows the results of NCI60 screening of Compound
6.
[0091] FIG. 25 shows the results of NCI60 screening of Compound
3.
[0092] FIG. 26 shows a schematic method of making a biotinylated
analog of Compound 27 (A) and the percent growth inhibition of
Compound 27 (B) and the biotinylated analog of Compound 27 (C).
[0093] FIG. 27 shows a schematic method of making a Compound 27
analog in which the phenyl ring is para substituted with an ethynyl
group.
[0094] FIG. 28 schematically shows a method of making a Compound 27
analog in which --CH.sub.2C.ident.CH is attached to the indole in
place of the phenyl ring.
[0095] FIG. 29 schematically shows a method of making a Compound 27
analog in which X is one and the substituent is
--O--CH.sub.2.ident.CH at the 5-indole position.
[0096] FIG. 30 schematically shows a method of making an acryl
Compound 27 analog in which --CH.sub.2Cl of the chloromethyl group
is replaced with an ethenyl group (A), and the percent growth
inhibition for the acryl analog (B).
[0097] FIG. 31 schematically shows a method of making a Compound 27
analog in which --CH.sub.2Cl of the chloromethyl group is replaced
with cyclopropoxyl.
[0098] FIG. 32 schematically shows a method of making a Compound 27
analog in which chloromethyl group is replaced with an oxoacetamide
group.
[0099] FIG. 33 schematically shows the cyclization of Compound 27
to produce the lactone.
[0100] FIG. 34 shows the percent growth inhibition of an analog of
Compound 27 in which an amide group and ethyl ester (diastereomer
data shown) replace the methyl ester.
[0101] FIG. 35 shows the percent growth inhibition of an analog of
Compound 27 in which the methyl benzoate is replaced with imidazole
(A) and pyridine (B).
[0102] FIG. 36 shows the percent growth inhibition of analogs of
Compound 27 in which the chloromethyl is replaced with
--O-tetrafluoromethyl (B), --OC(.dbd.O)CH.sub.3 (A) and --F
(C).
[0103] FIG. 37 shows a method of making an analog of Compound 27 in
which the methyl ester is replaced with propargyl.
[0104] FIG. 38 shows the percent growth inhibition of an analog of
Compound 27 in which methyl benzoate is replaced with phenyl.
[0105] FIG. 39 shows percent growth inhibition of an analog of
Compound 27 in which an alkyne acetate is substituted on the phenyl
ring.
[0106] FIG. 40 shows the percent growth vs. log concentration for
Compound 27 in the NCI60 test in all cell lines.
[0107] FIG. 41 shows a flow chart of the screening strategy for
identifying genetically selective compounds and ruling out
proliferation-dependent compounds.
[0108] FIG. 42 shows in panel A the effect of Compound 27 on
percent growth inhibition in four BJ-derived cell lines, and in
panel B the percent growth inhibition using alamar blue in a HT1080
cell line treated with Compound 27 in the presence of each
indicated bioactive compound.
[0109] FIG. 43 shows the percent growth inhibition using alamar
blue in HT1080 cells infected with lentivirus encoding shRNAs
targeting VDAC3 and treated with erastin in panel A, Compound 3 in
panel B, and Compound 27 in panel C.
[0110] FIG. 44 shows the mRNA levels (relative quantification) of
VDAC1, VDAC2, and VDAC3 to determine the specificity of shRNAs
targeting VDAC3 using Q-PCR.
[0111] FIG. 45 shows cell viability over time after treatment
determined from kinetic analysis of cell death induced by 5 ug/ml
erastin, 20 ug/ml Compound 3 and 0.5 ug/ml Compound 5 in panel A,
the time dependent effect of Compound 27 on three BJ cell lines on
growth inhibition in panel B, a photograph of the two BJ cell lines
treated with 5 ug/ml erastin, 20 ug/ml Compound 3, 0.5 .mu./mL of
Compound 27, or 1 .mu.M of staurosporine for 24 h. in panel C, and
lysates of staurosporine-treated but not erastin-treated, Compound
3-treated or Compound 27-treated BJ-TERT/LT/ST/RAS.sup.V12 cells
that contain the PARP-1 fragment produced by caspase-3/7 cleavage,
analyzed by Western blot with an antibody directed against human
PARP-1 in panel D.
[0112] FIG. 46 shows percent growth inhibition using alamar blue
indicating that Compound 27 was active in killing four KRAS
mutation harboring cancer cells, A549, Calu-1, PaCa-2 and HCT116
cells, treated for 24 hour in panel A; the activity of Compound 27
tested in 60 different human cancer cell lines where the left chart
shows the result of 18 cell lines that are representative of
sensitive and resistant groups (zero viability meaning a cytostatic
effect, and negative viability meaning a cytotoxic effect) and the
right table shows results of the COMPARE analysis for paclitaxel
and Compound 27 in panel B; percent growth inhibition using alamar
blue showing attenuation of RAS signaling in Calu-1 cells by
infecting with lentivirus containing shRNAs targeting H, N, or KRAS
confers resistance to Compound 27-induced lethality in panel C.
[0113] FIG. 47 shows percent growth inhibition in
staurosporine-treated cells to examine changes in drug
sensitivity.
[0114] FIG. 48 shows the results of flow cytometry to confirm iron
depletion of the iron chelators in BJ-TERT/LT/ST/RAS.sup.V12 cells
treated with the indicated iron chelators in panel A; percent
growth inhibition in BJ-TERT/LT/ST/RAS.sup.V12 cells treated with
Compound 27 in the presence or absence of iron chelators in panel
B; the level of TfR1 in each cell line was determined by Western
blot using an antibody against TfR1 using whole cell lysates from
BJ-TERT/LT/ST/RAS.sup.V12 cells infected with either non-targeting
shRNA (N.T.) or three different TfR1-targeting shRNAs (658, 659,
660) that were prepared, in panel C; the relative expression level
of a gene among the three cell lines was expressed as a ratio of
transcripts in a cell line to those in BJ-TERT where cellular RNAs
were prepared from each cell line, real-time PCR was performed with
each gene-specific primer set, and the expression levels of TfR1
(panel D), FTH1 (panel E) and FTL (panel F) were first normalized
to the level of endogenous control (RPLPO). FIG. 49 shows a
schematic method of making the 1R,3S and 1S,3S diastereomers of
Compound 27, formulas A and B, respectively.
[0115] FIG. 49 shows NMR data for the four stereoisomeric
conformations of Compound 27. Panel A shows NMR data for (1R,3S)
Compound 27. Panel B shows NMR data for (1S,3S) Compound 27. Panel
C shows NMR data for (1S,3R) Compound 27. Panel D shows NMR data
for (1R,3R) Compound 27. The NMR data indicate that the four
stereoisomers of Compound 27 were each enantiomerically pure. The
legend for the NMR data is as follows, stereochemistry not
shown.
##STR00025##
[0116] FIG. 50 shows NMR data for two of the stereoisomeric
conformations of Cyclized Compound 27. NMR data is shown for 1S,3S
Cyclized Compound 27 (panel A) and for 1R,3S Cyclized Compound 27
(panel B). The NMR data shows that these stereoisomers of Cyclized
Compound 27 were each enantiomerically pure. The legend for the NMR
data is as follows, stereochemistry not shown.
##STR00026##
[0117] FIG. 51 shows percent cell growth inhibition for the four
stereoisomeric conformations of Compound 27, namely (1R,3S)
Compound 27 (panel A), (1S,3S) Compound 27 (panel B), 1S,3R
Compound 27 (panel C), and 1R,3R Compound 27 (panel D), in
BJeH-hTERT (eH), BJeHLT-hTERT/LT/ST (eHLT),
BJeLR-hTERT/LT/ST/HRASG12V (eLR), and
BJDRD-hTERT/p53DD/CDK4/cyclinD1/ST/HRASG12V (DRD) cells. Only the
1S,3R Compound 27 stereoisomer demonstrates selective lethality
towards BJ tumor cell lines containing mutant HRAS versus isogenic
cell lines lacking oncogenic HRAS.
DETAILED DESCRIPTION OF THE INVENTION
[0118] Target-based, in-vitro screening has been successful for
developing cancer drugs as exemplified by Gleevec or Sorafenib.
Synthetic lethal screening using small molecules is a complementary
approach that involves testing compounds for selective lethality in
tumor cells harboring a specific oncogene. Synthetic lethal
screening using small molecules can identify cancer drug leads with
a potentially high therapeutic index. Oncogenic RAS (HRAS.sup.G12V)
is used as a specific mutation in synthetic lethal screening and
identified four compounds whose activity depends on
oncogenic-RAS-signaling. Mechanistic studies on these compounds
suggest that oncogenic-RAS-signaling in tumor cells enriches their
pool of iron, which is an important factor for enabling their
selective lethality. Further optimization of these compounds may
allow for the development of anticancer therapeutic agents with a
degree of genotype selectivity.
[0119] To circumvent concerns with the target-based approach in the
RAS field, the complementary approach of synthetic lethal screening
with small molecules was employed to identify those small molecules
that display synthetic lethality with oncogenic RAS. 47,725 small
molecules were tested in a series of isogenic cell lines. This
yielded four compounds with synthetic lethal interactions with
oncogenic RAS (HRAS.sup.G12V). Testing these four hit compounds in
multiple cell lines revealed proliferation-rate independent
activity, but increased lethality towards tumor cells with
oncogenic RAS. Counter-screening with chemical inhibitors to
identify suppressors of hit-compound-induced cell death revealed
some insights into their mechanism of action, such as MAPK/ERK
kinase (MEK) dependency and iron dependency. Cells transformed with
HRAS.sup.G12V have increased iron content relative to their normal
cell counterparts through up-regulation of the transferrin receptor
1.
[0120] The four hit compounds have synthetic lethal interactions
with oncogenic RAS (HRAS.sup.G12V), which suggests that the
activity of those compounds depends on HRAS.sup.G12V signaling.
Traditional chemotherapeutic reagents have shown high potency in
cancer cell killing, but they frequently target proteins directly
involved with DNA replication or metabolism, which makes them
proliferation-rate dependent, with a modest therapeutic index.
Considering this, lethal compounds with oncogenic-signal-dependent
activity have drawn interest as alternative chemotherapeutic
reagents.
[0121] Such iron-proliferation-dependent compounds were identified
by incorporating the DRD cell line into the counter-screening
process. Only hit compounds showing more potency in DRD cells than
that in BJ-TERT/LT/ST cells were chosen for further study. Hit
compounds that have almost the same growth inhibitory potency
between BJ-TERT/LT/ST/RAS.sup.V12 cells and DRD cells (Erastin,
Compound 3, and Compound 27) were shown to require activation of
MEK, a critical downstream effector of oncogenic RAS. Hit compounds
that have somewhat lower potency in DRD cells than in
BJ-TERT/LT/ST/RAS.sup.V12 cells (Compound 6 and Compound 3) showed
no dependency on MEK activation. Even with MEK-independent lethal
compounds, the contribution of HRAS.sup.G12V in increasing
sensitivity was observed suggesting that RAS downstream signals
other than the RAF-MEK-MAPK or the PI3K pathway are responsible for
the observed increase in sensitivity.
[0122] One interesting property of these compounds is their
iron-dependent mechanism of action. The iron dependency of these
compounds is evident because different iron chelators are able to
suppress the lethality induced by these compounds. Many cancer
cells are reported to have an enriched iron pool (Shterman et al.,
1991). Cancer patients have higher levels of iron than normal
individuals (Stevens et al., 1988). The BJ cell system recaptured
this relationship between iron and cancer, as
BJ-TERT/LT/ST/RAS.sup.V12 cells have more iron than their isogenic,
non-tumorigenic counterparts. The greater iron content in
BJ-TERT/LT/ST/RAS.sup.V12 cells, together with the iron-dependent
action of these hit compounds, suggests that the increased iron
pool within cancer cells might be a target for inducing
cancer-cell-specific lethality. Indeed, this possibility has been
explored by testing iron chelators for anticancer properties
(Kalinowski and Richardson, 2005). A number of iron chelators are
reported to have good potency in inhibiting cancer cell growth. For
example, bleomycin, a key component of standard chemotherapeutic
regimens for treating patient with germ-cell tumors (Kondagunta and
Motzer, 2006), is known to oxidatively damage DNA through its
complex with iron (Dorr, 1992). Another iron chelator, Triapine,
entered Phase II clinical trials in a combination therapy with
cisplatin to treat ovarian cancer (Low and Schoenfeldt, 2005).
[0123] There are two suggested mechanisms of lethality caused by
such iron chelators. One is that the iron chelators deplete iron
from critical biomolecules mediating the mitochondrial respiratory
chain (iron-sulfur proteins) and DNA synthesis (ribonucleotide
reductase), which leads to loss of cell viability (Cazzola et al.,
1990; Green et al., 2001). The other is that the iron chelators
generate reactive oxygen species by facilitating Fenton chemistry,
which damages biological molecules, such as nucleic acids, lipids,
and proteins (Halliwell and Gutteridge, 1990).
[0124] The compounds of the present invention do not deplete the
cellular iron pool. Therefore, it is unlikely that their lethality
stems from a similar mechanism of action of known iron chelators.
Instead, binding to target proteins may elicit iron-mediated toxic
responses involving either reactive oxygen species generation (in
case of Erastin and Compound 3) or not (in case of Compound 36 and
Compound 27). For example, the lethality of Erastin and Compound 3
was inhibited by both antioxidants and iron chelators. It is
possible that binding to target proteins causes a change in
cellular redox potential or a local redistribution of the iron pool
to initiate Fenton chemistry. Such an indirect role of iron in
anticancer drug-induced toxicity was suggested in a report
explaining iron-dependent lethality of anthracyclines (Kwok and
Richardson, 2003). Alternatively, binding to a target can
hyper-activate an oxidase complex, such as NADPH oxidase, to induce
a so-called "respiratory burst." The NADPH oxidase complex is found
to be up-regulated upon PDGF treatment exerting multiple effects on
carcinogenic processes (Arbiser et al., 2002). The NADPH oxidase
complex is also reported to be activated by some anticancer drugs,
such as paclitaxel (Alexandre et al., 2006) or arsenic acid (Chou
et al., 2004). The NADPH oxidase complex is essentially an electron
transport chain having iron-containing heme molecules (Cross and
Segal, 2004). Therefore, treatment with iron chelators removes iron
from the hemes, preventing the onset of a "respiratory burst" by a
compound.
[0125] The increased iron concentration in
BJ-TERT/LT/ST/RAS.sup.V12 cells compared to their isogenic counter
parts raises the possibility that oncogenic RAS signaling alters
iron metabolism to augment the cellular labile iron pool.
Oncogene-induced upregulation of cellular iron has been reported
for c-myc and E1A (Tsuji et al., 1995; Wu et al., 1999). Oncogenic
signals from c-myc or E1A downregulate expression of the heavy
subunit of ferritin (H-ferritin), which stores labile iron. Because
ferritin functions as an iron buffer, downregulation of ferritin by
oncogenic signals results in an increased intracellular iron pool.
Consequently, proliferation-driving enzymes, such as ribonucleotide
reductase, are replenished with sufficient iron.
[0126] In contrast, there are few established connections between
oncogenic RAS and iron metabolism. The existence of a
mitogen-responsive element in the 5' untranslated region (UTR) of
TfR1 mRNA has been reported, suggesting that RAS-RAF-MEK-MAPK
signaling may upregulate TfR1 level (Casey et al., 1988; Ouyang et
al., 1993). However, the data herein suggests that
oncogenic-RAS-signaling increases the cellular iron pool through
both TfR1-dependent and independent pathways. First, the expression
level of TfR1 in BJ-TERT, BJ-TERT/LT/ST, and
BJ-TERT/LT/ST/RAS.sup.V12 was gradually increased, while the total
amount of cellular iron abruptly increased in
BJ-TERT/LT/ST/RAS.sup.V12 cells, implying that the status of
iron-regulatory factors other than TfR1 was changed to accommodate
more iron. Second, diminishing TfR1 protein levels by shRNAs
resulted in only partial recovery of HT1080 cell death compared to
the result with iron chelators, which suggests that a
TfR1-independent labile iron pool that is regulated by
oncogenic-RAS-signaling contributed to the increase in cellular
iron and compound sensitivity. Efforts to identify target proteins
of these compounds may ultimately reveal novel proteins in iron
metabolism regulation; some such proteins are likely to be
connected to the RAS signaling pathway.
[0127] In summary, four novel lethal classes of compounds with
cancer-cell specificity have been identified using
isogonics-engineered cell lines and high-throughput cell-based
screening. While many currently used chemotherapeutic reagents are
cytostatic and proliferation-dependent in vitro, the compounds of
the present invention are cytotoxic and not
proliferation-dependent. Preliminary mechanism of action studies
with these compounds revealed that they have interesting features,
such as oncogenic-RAS-signal-dependent action and iron-dependent
activity. These results suggest a connection between oncogenic RAS
and iron metabolism, and support the notion that the cellular iron
pool is enriched by oncogenic signals and can be a target for
developing anti-cancer drugs with a favorable therapeutic
index.
[0128] In view of the foregoing, one embodiment of the present
invention is a compound having the formula I:
##STR00027##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.6 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl, wherein each alkyl, alkoxy, aralkyl, carbocyclic,
heterocyclic, aryl, heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl is optionally substituted with at least one
substituent; R.sub.4 and R.sub.5 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1$alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkynyl, aryl, carbocycle, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle, and
heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic may be optionally substituted
with at least one substituent; and X is 0-4 substituents on the
ring to which it is attached, or an enantiomer, optical isomer,
diastereomer, N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0129] All possible enantiomers, optical isomers, and diastomers of
each formula and compound recited herein are part of the invention,
whether they are explicitly shown or not. In the present invention,
the isomeric forms of the compounds may be synthesized de novo.
Alternatively, the specific desired isomeric form may be separated
from, e.g., a racemic solution using conventional techniques, such
as for example, gas chromatography. Moreover, the present
application includes every possible combination of each R group,
whether explicitly identified or not.
[0130] Preferably, in this embodiment, R.sub.2 is a substituted
aryl, a halo-substituted aryl, a tetra-substituted aryl, a
tetra-fluoro substituted aryl, or a C.sub.1-8 alkyl substituted
with carbonyl.
[0131] In certain embodiments, X represents 0-4 substituents. As
used herein, the term "substituent" means H, cyano, oxo, nitro,
acyl, acylamino, halogen, hydroxy, amino acid, amine, amide,
carbamate, ester, ether, carboxylic acid, thio, thioalkyl,
thioester, thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy,
C.sub.1-8alkenyl, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic,
3- to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, and arylsulfonyl. In certain embodiments X
represents one substituent, such as halogen or nitro, especially
chloro. In other embodiments, X represents no substituents on the
ring (i.e., all substituents are hydrogen atoms).
[0132] Preferred classes of compounds of formula I are defined
by:
##STR00028##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0133] One preferred compound of formula I is:
##STR00029##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0134] Another preferred compound of formula I is:
##STR00030##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0135] A further embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound of formula. II:
##STR00031##
wherein R.sub.1 and R.sub.2 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.3R.sup.4, OC(R.sup.3).sub.2COOH, SC(R.sup.3).sub.2COOH,
NHCHR.sup.3COOH, COR.sup.4, CO.sub.2R.sup.4, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.3R.sup.4,
OC(R.sup.3).sub.2COOH, SC(R.sup.3).sub.2COOH, NHCHR.sup.3COOH,
COR.sup.4, CO.sub.2R.sup.4, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.3 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.4 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; X is selected
from halo and C.sub.1-8 alkyl; and n is 0-8, or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0136] Preferably, each optional substituent is independently
selected from the group consisting of H, cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxilic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy, C.sub.1-8alkenyl,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, and arylsulfonyl.
[0137] In one preferred composition, the RAS-selective lethal
compound is a compound of formula IIa:
##STR00032##
or a compound of formula IIb:
##STR00033##
or enantiomers, optical isomers, diastereomers, N-oxides,
crystalline forms, hydrates, or pharmaceutically acceptable salts
of either formula IIa or IIb.
[0138] Representative, non-limiting examples of compounds of
formula II according to the present invention are compounds 3, 4,
and 5:
##STR00034##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0139] An additional embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound of formula III:
##STR00035##
wherein R.sub.1 is selected from H, halo, C.sub.1-8 alkyl,
C.sub.1-8alkylene, 1-hydroxyl-6-D-glucose optionally substituted
with from 1-4 acetates and/or 1-hydroxyl-6-D-glucose tetraacetate,
and 1-thionyl-.beta.-D-glucose optionally substituted with from 1-4
acetates, 0, and/or C.sub.1-8 alkoxy; R.sub.2 is selected from H,
halo, C.sub.1-8 alkyl, and 1-thionyl-.beta.-D-glucose substituted
with from 1-4 acetates, O, or C.sub.1-8 alkoxy; R.sub.3 and R.sub.4
are independently selected from H, halo, C.sub.1-8 alkyl, C.sub.1-8
alkoxy, 1-hydroxyl-.beta.-D-glucose optionally substituted with
from 1-4 acetates and/or 1-hydroxyl-.beta.-D-glucose tetraacetate,
and 1-thionyl-.beta.-D-glucose optionally substituted with from 1-4
acetates; R.sub.5 and R.sub.6 are independently selected from H,
OH, and acetate esters; and R.sub.7 is selected from H or carbonyl,
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0140] Preferably, the RAS-selective lethal compound is a compound
of formula IIIa:
##STR00036##
wherein n is 0-8, or an enantiomer, optical isomer, diastereomer,
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof. Preferably, n is 1-8. In the present invention, where
a numerical range is described, all integers within the range,
including the end points are intended to be included.
[0141] Representative, non-limiting examples of compounds for
formula III according to the present invention include compounds
6-26:
##STR00037## ##STR00038## ##STR00039## ##STR00040##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0142] Another embodiment of the present invention is a composition
containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula IV:
##STR00041##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
are independently selected from H, Halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkynyl, aryl, carbocycle, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle, and
heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic may be optionally substituted
with at least one substituent; and X is 0-4 substituents on the
ring to which it is attached, or an enantiomer, optical isomer,
diastereomer, N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0143] Preferably, each optional substituent is independently
selected from the group consisting of H, cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxilic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy, C.sub.1-8alkenyl,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, and arylsulfonyl.
[0144] Preferably, the RAS-selective lethal compound is a compound
of formula IVa, IVb, IVc, or IVd:
##STR00042##
[0145] Representative, non-limiting examples of compounds of
formula IV according to the present invention include compounds
27-35:
##STR00043## ##STR00044## ##STR00045##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0146] A further embodiment of the present invention is a
composition containing a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula V:
##STR00046##
wherein R.sub.1 is independently selected from H, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.2R.sup.3,
OC(R.sup.2).sub.2COOH, SC(R.sup.2).sub.2COOH, NHCHR.sup.2COOH,
COR.sup.3, CO.sub.2R.sup.3, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.2R.sup.3,
OC(R.sup.2).sub.2COOH, SC(R.sup.2).sub.2COOH, NHCHR.sup.2COOH,
COR.sup.3, CO.sub.2R.sup.3, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.2 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.3 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and
A is C, N, or S, or
[0147] an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0148] Preferably, each optional substituent is independently
selected from the group consisting of H, cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxilic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy, C.sub.1-8alkenyl,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, and arylsulfonyl.
[0149] Representative, non-limiting examples of compounds of
formula V according to the present invention include compounds 36
and 37:
##STR00047##
or an enantiomer, optical isomer, diastereomer, N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0150] Another embodiment of the present invention is a compound
having the formula VI:
##STR00048##
wherein R.sub.2 and R.sub.3 are independently selected from H,
methyl, methyl benzoate, propargyl, and phenyl, wherein at least
one of R.sub.2 and R.sub.3 is other than H; R.sub.6, R.sub.8, and
R.sub.9 are independently selected from H, halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached; or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0151] In one aspect of this embodiment, the compound of formula VI
has an optional substituent, which is independently selected from
the group consisting of H, cyano, oxo, nitro, acyl, acylamino,
halogen, hydroxy, amino acid, amine, amide, carbamate, ester,
ether, carboxylic acid, thio, thioalkyl, thioester, thioether,
C.sub.1-8alkyl, C.sub.1-8alkoxy, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne, C.sub.1-8
alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0152] A preferable embodiment is a compound of formula VI in which
R.sub.2 is phenyl and R.sub.3 is H.
[0153] Another preferable embodiment is a compound of formula VI,
wherein R.sub.2 is methyl benzoate and R.sub.3 is H.
[0154] Another preferable embodiment is a compound of formula VI,
wherein R.sub.2 and R.sub.3 are each methyl.
[0155] In another embodiment of the present invention, a compound
is provided having the formula VII:
##STR00049##
wherein R.sub.2 is selected from halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether, wherein each
alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sub.5,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or an enantiomer,
optical isomer, diastereomer, N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof.
[0156] In one aspect of this embodiment, the compound of formula
VII has an optional substituent, which is independently selected
from the group consisting of cyano, oxo, nitro, acyl, acylamino,
halogen, hydroxy, amino acid, amine, amide, carbamate, ester,
ether, carboxylic acid, thio, thioalkyl, thioester, thioether,
C.sub.1-8 alkyl, C.sub.1-8alkene, C.sub.1-8alkyne, C.sub.1-8alkoxy,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne, C.sub.1-8
alkyl alkyne and C.sub.1-8 alkyne acetate.
[0157] A preferred embodiment of a compound according to formula
VII, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0158] A preferred embodiment of a compound according to formula
VII is wherein R.sub.2 is --(CH.sub.2).sub.1-8--C.ident.CH.
[0159] A preferred embodiment of a compound according to formula
VII is wherein R.sub.2 is --(CH.sub.2)C.ident.CH and X is zero.
[0160] Another preferred embodiment of a compound according to
formula VII is wherein X is 1-4 substituents, and at least one
substituent for X is --O(CH.sub.2).sub.1-8--C.ident.CH.
[0161] Another preferred embodiment of a compound according to
formula VII is wherein X is 1 substituent and the substituent for X
is --O(CH.sub.2)--C.ident.CH at the 5-indole position, and R.sub.3
is methyl benzoate.
[0162] A further embodiment of the present invention is a compound
having the formula VIII:
##STR00050##
wherein R.sub.2 and R.sub.3 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether and is
optionally substituted with at least one substituent, and wherein
at least one of R.sub.2 and R.sub.3 is other than H; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; n is 0 or 1; E is
an electrophilic group that has a lower electrophilicity index
value than does Cl; or an enantiomer, optical isomer, diastereomer,
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof.
[0163] In one aspect of this embodiment, the compound according to
formula VIII has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8alkyl, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne,
C.sub.1-8 alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0164] A preferred embodiment of the compound according to formula
VIII, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0165] A preferred embodiment of the compound according to formula
VIII, is wherein n is zero.
[0166] Another preferred embodiment of the compound according to
formula VIII, is wherein E is selected from the group consisting of
cyclopropoxyl; --C(.dbd.O)H; N.sub.3; --OC(.dbd.O)CH.sub.3; and
--O-tetrafluorophenyl.
[0167] A further embodiment of the compound according to formula
VIII, is wherein n is zero.
[0168] Another embodiment of the compound according to formula
VIII, is wherein E is an electrophilic group selected from the
group consisting of Br, I, hypochlorite, sulfur dioxide, carbon
disulfide, benzene, and sodium.
[0169] A preferred embodiment of the compound according to formula
VIII, is wherein R.sub.2 is --(CH.sub.2).sub.1-8--C.ident.CH.
[0170] A preferred embodiment of the compound according to formula
VIII, is wherein X is 1-4 substituents, and at least one
substituent for X is --O(CH.sub.2).sub.1-8--C.ident.CH.
[0171] A further embodiment of the present invention is a compound
according to formula IX:
##STR00051##
wherein R.sub.6, R.sub.8, and R.sub.9 are independently selected
from H, halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; and R.sub.10 is
selected from the group consisting of a C.sub.1-8 ester and an
acid, which ester or acid is hydrolyzable in vivo; or an
enantiomer, optical isomer, diastereomer, N-oxide, crystalline
form, hydrate, or pharmaceutically acceptable salt thereof.
[0172] In one aspect of this embodiment, the compound according to
formula IX, has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8alkyl, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne,
C.sub.1-8 alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0173] A preferred embodiment of a compound according to formula
IX, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0174] A preferred embodiment of a compound according to formula
IX, is wherein R.sub.10 is --COOH or --C(.dbd.O)OCH.sub.3.
[0175] As noted previously, it has been found that some of the
compounds exhibit stereoisomeric selectivity in in vitro biological
assays. Indeed, as shown in more detail below, surprisingly, in
certain cases only one of, e.g., four stereoisomers of a particular
compound are active in the in vitro activity experiments disclosed
herein. Such stereoisomeric selectivity in this area is highly
unusual and could not have been predicted.
[0176] In view of the foregoing, another embodiment of the present
invention is a compound having the formula X:
##STR00052##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.6 are independently
selected from H, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl, wherein each alkyl, alkoxy, aralkyl, carbocyclic,
heterocyclic, aryl, heteroaryl, acyl, alkylsulfonyl, and
arylsulfonyl is optionally substituted with at least one
substituent; R.sub.4 and R.sub.5 are independently selected from H,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, C.sub.1-8alkyl, carbocycle, aryl, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle,
wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally
substituted with at least one substituent; R.sup.8 is selected from
H, C.sub.1-8alkyl, C.sub.1-8alkenyl, C.sub.1-8alkynyl, aryl,
carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl,
alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl,
alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, alkylheterocycle, and heteroaromatic may be
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline form,
hydrate, or pharmaceutically acceptable salt thereof.
[0177] In a preferred embodiment, R.sub.2 is a larger and/or
bulkier group than is R.sub.3. A preferable R.sub.3 is H.
[0178] Preferable embodiments are compounds of formula X in which
R.sub.2 is a substituted aryl, a halo-substituted aryl, a
tetra-substituted aryl, a tetra-fluoro substituted aryl, or a
C.sub.1-8 alkyl substituted with carbonyl.
[0179] In certain embodiments, substituent X represents 0-4
substituents. As used in such embodiments, the term "substituent"
means H, cyano, oxo, nitro, acyl, acylamino, halogen, hydroxy,
amino acid, amine, amide, carbamate, ester, ether, carboxylic acid,
thio, thioalkyl, thioester, thioether, C.sub.1-8 alkyl,
C.sub.1-8alkoxy, C.sub.1-8alkenyl, C.sub.1-8aralkyl, 3- to
8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, sulfate,
sulfonamide, sulfoxide, sulfonate, sulfone, alkylsulfonyl, and
arylsulfonyl. In certain embodiments, substituent X represents one
substituent, such as halogen or nitro, especially chloro. In other
embodiments, substituent X represents no substituents on the ring
(i.e., all substituents are hydrogen atoms).
[0180] In certain embodiments, substituents of Formula X have an
optional substituent. Preferably, each optional substituent is
independently selected from the group consisting of H, cyano, oxo,
nitro, acyl, acylamino, halogen, hydroxy, amino acid, amine, amide,
carbamate, ester, ether, carboxilic acid, thio, thioalkyl,
thioester, thioether, C.sub.1-8 alkyl, C.sub.1-8alkoxy,
C.sub.1$alkenyl, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, and arylsulfonyl.
[0181] Preferred classes of compounds of formula X are defined
by:
##STR00053##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0182] A further embodiment of the invention is a compound selected
from the group consisting of:
##STR00054##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0183] Preferably, the compound of this embodiment is (1S,3R)
Compound 27 (shown as formula C above), or a N-oxide, crystalline
form, hydrate, or pharmaceutically acceptable salt thereof. More
preferably, (1S,3R) Compound 27 is enantiomerically pure.
[0184] A preferable embodiment of the present invention is an
enantiomerically pure compound having the structure:
##STR00055##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0185] One preferred compound of the present invention is:
##STR00056##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0186] Another preferred compound of the present invention is:
##STR00057##
wherein the phenyl ring is optionally substituted with
methoxycarbonyl, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0187] Another embodiment of the present invention is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a RAS-selective lethal compound according to any one of
formulas X, Xa, Xb, X1 and X2 and stereoisomers of Compound 27 of
formulas A, B, C, and D, or a N-oxide, crystalline form, hydrate,
or pharmaceutically acceptable salt thereof. When the
pharmaceutical composition comprises a stereoisomer of Compound 27,
preferably a stereoisomer of formula C is present.
[0188] A preferred embodiment is a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an
enantiomerically pure RAS-selective lethal compound having the
structure:
##STR00058##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0189] Another embodiment of the present invention is a composition
comprising a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to formula XI:
##STR00059##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
are independently selected from H, Halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sup.7 is
selected from H, carbocycle, aryl, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, and alkylheterocycle, wherein each
alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl,
alkylheteroaryl, and alkylheterocycle may be optionally substituted
with at least one substituent; R.sup.8 is selected from H,
C.sub.1-8alkynyl, aryl, carbocycle, heteroaryl, heterocycle,
alkylaryl, alkylheteroaryl, alkylheterocycle, and heteroaromatic,
wherein each alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl,
heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle, and
heteroaromatic may be optionally substituted with at least one
substituent; and X is 0-4 substituents on the ring to which it is
attached, wherein R.sub.2 and R.sub.3 are not the same group, or a
N-oxide, crystalline form, hydrate, or pharmaceutically acceptable
salt thereof.
[0190] In a preferred embodiment, R.sub.2 is a larger and/or
bulkier group than is R.sub.3. A preferable R.sub.3 is H.
[0191] In one aspect of this embodiment, the compound of formula XI
has an optional substituent, which is independently selected from
the group consisting of H, cyano, oxo, nitro, acyl, acylamino,
halogen, hydroxy, amino acid, amine, amide, carbamate, ester,
ether, carboxilic acid, thio, thioalkyl, thioester, thioether,
C.sub.1-8 alkyl, C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to
8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, sulfate,
sulfonamide, sulfoxide, sulfonate, sulfone, alkylsulfonyl, and
arylsulfonyl.
[0192] In this embodiment, the RAS-selective lethal compound of the
composition is a compound of formula XIa:
##STR00060##
[0193] Representative, non-limiting examples of compounds of
formula XI present in the composition include the following:
##STR00061## ##STR00062## ##STR00063##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0194] Another embodiment of the present invention is a compound
having the formula XII:
##STR00064##
wherein R.sub.2 and R.sub.3 are independently selected from H,
methyl, methyl benzoate, propargyl, and phenyl, wherein at least
one of R.sub.2 and R.sub.3 is other than H; R.sub.6, R.sub.8, and
R.sub.9 are independently selected from H, halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein
each alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached; wherein R.sub.2
and R.sub.3 are not the same group, or a N-oxide, crystalline
forth, hydrate, or pharmaceutically acceptable salt thereof.
[0195] A preferred embodiment is a compound of formula XII in which
R.sub.2 is a larger and/or bulkier group than is R.sub.3. A
preferable R.sub.3 is H. And, substituent X is as previously
defined.
[0196] In one aspect of this embodiment, the compound of formula
XII has an optional substituent, which is independently selected
from the group consisting of H, cyano, oxo, nitro, acyl, acylamino,
halogen, hydroxy, amino acid, amine, amide, carbamate, ester,
ether, carboxylic acid, thio, thioalkyl, thioester, thioether,
C.sub.1-8alkyl, C.sub.1-8alkoxy, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne, C.sub.1-8
alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0197] A preferable embodiment is a compound of formula XII in
which R.sub.2 is phenyl and R.sub.3 is H.
[0198] Another preferable embodiment is a compound of formula XII,
wherein R.sub.2 is methyl benzoate and R.sub.3 is H.
[0199] Another embodiment of the present invention is a compound
having the formula XIII:
##STR00065##
wherein R.sub.2 is selected from halo, C.sub.1-8alkyl,
C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether, wherein each
alkyl, alkoxy, carbocyclic, heterocyclic, aryl, heteroaryl,
carboxylate, ester, ether, amide, carbohydrate, amino acid, acyl,
alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; and X is 0-4
substituents on the ring to which it is attached, or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0200] In one aspect of this embodiment, the compound according to
formula XIII has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8 alkyl, C.sub.1-8alkyne, C.sub.1-8aralkyl, 3-
to 8-membered carbocyclic, 3- to 8-membered heterocyclic, 3- to
8-membered aryl, or 3- to 8-membered heteroaryl, sulfate,
sulfonamide, sulfoxide, sulfonate, sulfone, alkylsulfonyl,
arylsulfonyl, C.sub.1-8alkoxy alkyne, C.sub.1-8 alkyl alkyne and
C.sub.1-8 alkyne acetate. And, substituent X is as previously
defined.
[0201] A preferred embodiment of a compound according to formula
XIII, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0202] Another preferred embodiment of a compound according to
formula XIII, is wherein R.sub.2 is
--(CH.sub.2).sub.1-8C.ident.CH.
[0203] A further preferred embodiment of a compound according to
formula XIII, is wherein R.sub.2 is --(CH.sub.2)C.ident.CH and X is
zero.
[0204] Another preferred embodiment of a compound according to
formula XIII, is wherein X is 1-4 substituents, and at least one
substituent for X is --O(CH.sub.2).sub.1-8--C.ident.CH.
[0205] Another preferred embodiment of a compound according to
formula XIII, is wherein X is 1 substituent and the substituent for
X is --O(CH.sub.2)--C.ident.CH at the 5-indole position, and
R.sub.2 is methyl benzoate.
[0206] A further embodiment of the present invention is a compound
having the formula XIV:
##STR00066##
wherein R.sub.2 and R.sub.3 are independently selected from H,
halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl; alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, thioether and is
optionally substituted with at least one substituent, and wherein
at least one of R.sub.2 and R.sub.3 is other than H; R.sub.6,
R.sub.8, and R.sub.9 are independently selected from H, halo,
C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, carboxylate, ester, amide, carbohydrate,
amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid,
acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; n is 0 or 1; E is
an electrophilic group that has a lower electrophilicity index
value than does Cl; wherein R.sub.2 and R.sub.3 are not the same
group, or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0207] A preferred embodiment is a compound of formula XIV in which
R.sub.2 is a larger and/or bulkier group than is R.sub.3. A
preferable R.sub.3 is H. And, substituent X is as previously
defined.
[0208] In one aspect of this embodiment, the compound according to
formula XIV has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1-8alkyl, C.sub.1-8alkene, C.sub.1-8alkyne,
C.sub.1-8alkoxy, C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3-
to 8-membered heterocyclic, 3- to 8-membered aryl, or 3- to
8-membered heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate,
sulfone, alkylsulfonyl, arylsulfonyl, C.sub.1-8alkoxy alkyne,
C.sub.1-8 alkyl alkyne, and C.sub.1-8 alkyne acetate.
[0209] A preferred embodiment of the compound according to formula
XIV, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0210] Another preferred embodiment of the compound according to
formula XIV, is wherein n is zero.
[0211] Another preferred embodiment of the compound according to
formula XIV, is wherein E is selected from the group consisting of
cyclopropoxyl; --C(.dbd.O)H; N.sub.3; --OC(.dbd.O)CH.sub.3; and
--O-tetrafluorophenyl.
[0212] A further preferred embodiment of the compound according to
formula XIV, is wherein n is zero.
[0213] Another preferred embodiment of the compound according to
formula XIV, is wherein E is an electrophilic group selected from
the group consisting of Br, I, hypochlorite, sulfur dioxide, carbon
disulfide, benzene, and sodium.
[0214] Another preferred embodiment of the compound according to
formula XIV, is wherein R.sub.2 is
(CH.sub.2).sub.1-8--C.ident.CH.
[0215] Another preferred embodiment of the compound according to
formula XIV, is wherein X is 1-4 substituents, and at least one
substituent for X is --O(CH.sub.2).sub.1-8--C.ident.CH.
[0216] A further embodiment of the present invention is a compound
according to formula XV:
##STR00067##
wherein R.sub.6, R.sub.8, and R.sub.9 are independently selected
from H, halo, C.sub.1-8alkyl, C.sub.1-8alkoxy, 3- to 8-membered
carbocyclic, 3- to 8-membered heterocyclic, 3- to 8-membered aryl,
or 3- to 8-membered heteroaryl, carboxylate, ester, ether, amide,
carbohydrate, amino acid, acyl, alkoxy-substituted acyl, alditol,
NR.sup.7R.sup.8, OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH,
NHCHR.sup.7COOH, COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide,
sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether,
wherein each alkyl, alkoxy, carbocyclic, heterocyclic, aryl,
heteroaryl, carboxylate, ester, ether, amide, carbohydrate, amino
acid, acyl, alkoxy-substituted acyl, alditol, NR.sup.7R.sup.8,
OC(R.sup.7).sub.2COOH, SC(R.sup.7).sub.2COOH, NHCHR.sup.7COOH,
COR.sup.8, CO.sub.2R.sup.8, sulfate, sulfonamide, sulfoxide,
sulfonate, sulfone, thioalkyl, thioester, and thioether is
optionally substituted with at least one substituent; X is 0-4
substituents on the ring to which it is attached; and R.sub.10 is
selected from the group consisting of a C.sub.1-8 ester and an
acid, which ester or acid is hydrolyzable in vivo; or a N-oxide,
crystalline form, hydrate, or pharmaceutically acceptable salt
thereof.
[0217] In one aspect of this embodiment, the compound according to
formula XV has an optional substituent, which is independently
selected from the group consisting of cyano, oxo, nitro, acyl,
acylamino, halogen, hydroxy, amino acid, amine, amide, carbamate,
ester, ether, carboxylic acid, thio, thioalkyl, thioester,
thioether, C.sub.1$alkyl, C.sub.1-8alkene, C.sub.1-8alkoxy,
C.sub.1-8aralkyl, 3- to 8-membered carbocyclic, 3- to 8-membered
heterocyclic, 3- to 8-membered aryl, or 3- to 8-membered
heteroaryl, sulfate, sulfonamide, sulfoxide, sulfonate, sulfone,
alkylsulfonyl, arylsulfonyl, C.sub.1-10alkoxy alkyne, C.sub.1-8
alkyl alkyne, and C.sub.1-8 alkyne acetate. And, substituent X is
as previously defined.
[0218] A preferred embodiment of a compound according to formula
XV, is wherein R.sub.6, R.sub.8, and R.sub.9 are each H.
[0219] Another preferred embodiment of a compound according to
formula XV, is wherein R.sub.10 is --COOH or
--C(.dbd.O)OCH.sub.3.
[0220] In another aspect of the present invention, a compound is
provided which is selected from the group consisting of:
##STR00068##
and mixtures thereof, or a N-oxide, crystalline form, hydrate, or
pharmaceutically acceptable salt thereof.
[0221] In this embodiment, preferably the compound is:
##STR00069##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0222] A more preferred embodiment is wherein the compound is
enantiomerically pure.
[0223] A further preferred embodiment is an enantiomerically pure
compound having the structure:
##STR00070##
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0224] Compositions, including pharmaceutical compositions are
provided comprising a pharmaceutically acceptable carrier and a
RAS-selective lethal compound according to any one of formulas XV
and stereoisomers of cyclic compound 27 of formulas E, F, G, and H,
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof. When the pharmaceutical composition
comprises a stereoisomer of Cyclized Compound 27, preferably a
stereoisomer of formula G is present.
[0225] A preferred embodiment of the present invention is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an enantiomerically pure RAS-selective lethal compound
having the structure:
##STR00071##
[0226] or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0227] It is contemplated that all embodiments of the invention can
be combined with one or more other embodiments, even those
described under different aspects of the invention.
[0228] Compositions, including pharmaceutical compositions are
provided comprising, optionally a pharmaceutical carrier, and a
compound selected from the group consisting of compounds of formula
VI, compounds of formula VII, compounds of formula VIII, and
compounds of formula IX, which compounds are RAS-selective lethal
compounds.
[0229] Compositions, including pharmaceutical compositions are
provided comprising a RAS-selective lethal compound according to
any one of formulas X, Xa, Xb, X1, X2, XII, XIII, XIV, XV,
stereoisomers of Compound 27 of formulas A, B, C, and D,
stereoisomers of Cyclized Compound 27 of formulas E, F, G, and H,
or a N-oxide, crystalline form, hydrate, or pharmaceutically
acceptable salt thereof.
[0230] As used herein, the term "acyl" has its art-recognized
meaning and refers to a group represented by the general formula
hydrocarbylC(O)--, preferably alkylC(O)--.
[0231] As used herein, the term "acylamino" has its art-recognized
meaning and refers to an amino group substituted with an acyl group
and may be represented, for example, by the formula
hydrocarbylC(O)NH--.
[0232] As used herein, the term "acyloxy" has its art-recognized
meaning and refers to a group represented by the general formula
hydrocarbylC(O)O--, preferably alkylC(O)O--.
[0233] The term "alkoxy" refers to an alkyl group, preferably a
lower alkyl group, having an oxygen attached thereto.
Representative alkoxy groups include methoxy, ethoxy, propoxy,
tert-butoxy and the like. Other alkoxy groups within the scope of
the invention include, for example, the following:
##STR00072##
[0234] The term "alkoxyalkyl" refers to an alkyl group substituted
with an alkoxy group and may be represented by the general formula
alkyl-O-alkyl.
[0235] The term "alkenyl", as used herein, refers to an aliphatic
group containing at least one double bond and is intended to
include both "unsubstituted alkenyls" and "substituted alkenyls",
the latter of which refers to alkenyl moieties having substituents
replacing a hydrogen on one or more carbons of the alkenyl group.
Such substituents may occur on one or more carbons that are
included or not included in one or more double bonds. Moreover,
such substituents include all those contemplated for alkyl groups,
as discussed below, except where stability is prohibitive. For
example, substitution of alkenyl groups by one or more alkyl,
carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is
contemplated.
[0236] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups,
alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted
alkyl groups. In preferred embodiments, a straight chain or
branched chain alkyl has 30 or fewer carbon atoms in its backbone
(e.g., C.sub.1-C.sub.30 for straight chains, C.sub.3-C.sub.30 for
branched chains), and more preferably 20 or fewer, such as from 1
to 8. Likewise, preferred cycloalkyls have from 3-10 carbon atoms
in their ring structure, and more preferably have 5, 6 or 7 carbons
in the ring structure.
[0237] Moreover, unless otherwise indicated, the term "alkyl" (or
"lower alkyl") as used throughout the specification, examples, and
claims is intended to include both "unsubstituted alkyls" and
"substituted alkyls", the latter of which refers to alkyl moieties
having substituents replacing a hydrogen on one or more carbons of
the hydrocarbon backbone. Such substituents can include, for
example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an
alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a
thioester, a thioacetate, or a thioformate), an alkoxyl, a
phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an
amido, an amidine, an imine, a cyano, a nitro, an azido, a
sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a
sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic
or heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. For instance, the
substituents of a substituted alkyl may include substituted and
unsubstituted forms of amino, azido, imino, amido, phosphoryl
(including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups,
as well as ethers, alkylthios, carbonyls (including ketones,
aldehydes, carboxylates, and esters), --CF.sub.3, --CN and the
like. Exemplary substituted alkyls are described below. Cycloalkyls
can be further substituted with alkyls, alkenyls, alkoxys,
alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF.sub.3,
--CN, and the like.
[0238] The term "C.sub.x-y" when used in conjunction with a
chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl,
or alkoxy is meant to include groups that contain from x to y
carbons in the chain. For example, the term "C.sub.x-yalkyl" refers
to substituted or unsubstituted saturated hydrocarbon groups,
including straight-chain alkyl and branched-chain alkyl groups that
contain from x to y carbons in the chain, including haloalkyl
groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.
C.sub.0 alkyl indicates a hydrogen where the group is in a terminal
position, a bond if internal. The terms "C.sub.2-yalkenyl" and
"C.sub.2-yalkynyl" refer to substituted or unsubstituted
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls described above, but that contain at
least one double or triple bond respectively.
[0239] The term "alkylamino", as used herein, refers to an amino
group substituted with at least one alkyl group.
[0240] The term "alkylthio", as used herein, refers to a thiol
group substituted with an alkyl group and may be represented by the
general formula alkylS--.
[0241] The term "alkynyl", as used herein, refers to an aliphatic
group containing at least one triple bond and is intended to
include both "unsubstituted alkynyls" and "substituted alkynyls",
the latter of which refers to alkynyl moieties having substituents
replacing a hydrogen on one or more carbons of the alkynyl group.
Such substituents may occur on one or more carbons that are
included or not included in one or more triple bonds. Moreover,
such substituents include all those contemplated for alkyl groups,
as discussed above, except where stability is prohibitive. For
example, substitution of alkynyl groups by one or more alkyl,
carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is
contemplated.
[0242] The term "amide", as used herein, refers to a group
##STR00073##
[0243] wherein R.sup.7 and R.sup.8 each independently represent a
hydrogen or hydrocarbyl group, or R.sup.7 and R.sup.8 taken
together with the N atom to which they are attached complete a
heterocycle having from 4 to 8 atoms in the ring structure.
[0244] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines and salts thereof,
e.g., a moiety that can be represented by
##STR00074##
[0245] wherein R.sup.7, R.sup.8, and R.sup.8' each independently
represent a hydrogen or a hydrocarbyl group, or R.sup.7 and R.sup.8
taken together with the N atom to which they are attached complete
a heterocycle having from 4 to 8 atoms in the ring structure.
[0246] The term "aminoalkyl", as used herein, refers to an alkyl
group substituted with an amino group.
[0247] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group.
[0248] The term "aryl" as used herein include substituted or
unsubstituted single-ring aromatic groups in which each atom of the
ring is carbon. Preferably the ring is a 3- to 8-membered ring,
more preferably a 6-membered ring. The term "aryl" also includes
polycyclic ring systems having two or more cyclic rings in which
two or more carbons are common to two adjoining rings wherein at
least one of the rings is aromatic, e.g., the other cyclic rings
can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,
heteroaryls, and/or heterocyclyls. Aryl groups include benzene,
naphthalene, phenanthrene, phenol, aniline, and the like.
[0249] The term "carbamate" is art-recognized and refers to a
group
##STR00075##
wherein R.sup.7 and R.sup.8 independently represent hydrogen or a
hydrocarbyl group.
[0250] The terms "carbocycle", "carbocyclyl", and "carbocyclic", as
used herein, refers to a non-aromatic saturated or unsaturated ring
in which each atom of the ring is carbon. Preferably a carbocycle
ring contains from 3 to 10 atoms, more preferably from 3 to 8
atoms, including 5 to 7 atoms, such as for example, 6 atoms.
[0251] The term "carbocyclylalkyl", as used herein, refers to an
alkyl group substituted with a carbocycle group.
[0252] The term "carbonate" is art-recognized and refers to a group
--OCO.sub.2--R.sup.7, wherein R.sup.7 represents a hydrocarbyl
group.
[0253] The term "carboxy", as used herein, refers to a group
represented by the formula --CO.sub.2H.
[0254] The term "ester", as used herein, refers to a group
--C(O)OR.sup.7 wherein R.sup.7 represents a hydrocarbyl group.
[0255] The term "ether", as used herein, refers to a hydrocarbyl
group linked through an oxygen to another hydrocarbyl group.
Accordingly, an ether substituent of a hydrocarbyl group may be
hydrocarbyl-O--. Ethers may be either symmetrical or unsymmetrical.
Examples of ethers include, but are not limited to,
heterocycle-.beta.-heterocycle and aryl-O-heterocycle. Ethers
include "alkoxyalkyl" groups, which may be represented by the
general formula alkyl-O-alkyl.
[0256] The terms "halo" and "halogen" are used interchangeably
herein and mean halogen and include chloro, fluoro, bromo, and
iodo.
[0257] The terms "hetaralkyl" and "heteroaralkyl", as used herein,
refers to an alkyl group substituted with a hetaryl group.
[0258] The terms "heteroaryl" and "hetaryl" include substituted or
unsubstituted aromatic single ring structures, preferably 3- to
8-membered rings, more preferably 5- to 7-membered rings, even more
preferably 5- to 6-membered rings, whose ring structures include at
least one heteroatom, preferably one to four heteroatoms, more
preferably one or two heteroatoms. The terms "heteroaryl" and
"hetaryl" also include polycyclic ring systems having two or more
cyclic rings in which two or more carbons are common to two
adjoining rings wherein at least one of the rings is
heteroaromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or
heterocyclyls. Heteroaryl groups include, for example, pyrrole,
furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine,
pyrazine, pyridazine, and pyrimidine, and the like.
[0259] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, and sulfur.
[0260] The terms "heterocyclyl", "heterocycle", and "heterocyclic"
refer to substituted or unsubstituted non-aromatic ring structures,
preferably 3- to 10-membered rings, more preferably 3- to
8-membered rings, whose ring structures include at least one
heteroatom, preferably one to four heteroatoms, more preferably one
or two heteroatoms. The terms "heterocyclyl" and "heterocyclic"
also include polycyclic ring systems having two or more cyclic
rings in which two or more carbons are common to two adjoining
rings wherein at least one of the rings is heterocyclic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heterocyclyl groups include, for example, piperidine, piperazine,
pyrrolidine, morpholine, lactones, lactams, and the like.
[0261] The term "heterocyclylalkyl", as used herein, refers to an
alkyl group substituted with a heterocycle group.
[0262] The term "hydrocarbyl", as used herein, refers to a group
that is bonded through a carbon atom that does not have a .dbd.O or
.dbd.S substituent, and typically has at least one carbon-hydrogen
bond and a primarily carbon backbone, but may optionally include
heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and
trifluoromethyl are considered to be hydrocarbyl for the purposes
of this application, but substituents such as acetyl (which has a
.dbd.O substituent on the linking carbon) and ethoxy (which is
linked through oxygen, not carbon) are not. Hydrocarbyl groups
include, but are not limited to aryl, heteroaryl, carbocycle,
heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.
[0263] The term "hydroxyalkyl", as used herein, refers to an alkyl
group substituted with a hydroxy group.
[0264] The term "lower" when used in conjunction with a chemical
moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy
is meant to include groups where there are ten or fewer
non-hydrogen atoms in the substituent, preferably eight or fewer,
such as for example, from about 3 to 8 carbon atoms, more
preferably less than 6 carbon atoms. A "lower alkyl", for example,
refers to an alkyl group that contains ten or fewer carbon atoms,
preferably six or fewer. In certain embodiments, acyl, acyloxy,
alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are
respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl,
lower alkynyl, or lower alkoxy, whether they appear alone or in
combination with other substituents, such as in the recitations
hydroxyalkyl and aralkyl (in which case, for example, the atoms
within the aryl group are not counted when counting the carbon
atoms in the alkyl substituent).
[0265] The terms "polycyclyl", "polycycle", and "polycyclic" refer
to two or more rings (e.g., cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which
two or more atoms are common to two adjoining rings, e.g., the
rings are "fused rings". Each of the rings of the polycycle can be
substituted or unsubstituted. In certain embodiments, each ring of
the polycycle contains from 3 to 10 atoms in the ring, preferably
from 3 to 8, such as for example, 5 to 7.
[0266] The term "substituted" refers to moieties having
substituents replacing a hydrogen on one or more carbons of the
backbone. It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc. As used
herein, the term "substituted" is contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
non-aromatic substituents of organic compounds. The permissible
substituents can be one or more and the same or different for
appropriate organic compounds. For purposes of this invention, the
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
which satisfy the valences of the heteroatoms. Substituents can
include any substituents described herein, for example, a halogen,
a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphate, a phosphonate, a phosphinate, an amino, an amido, an
amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an
alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a
sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate.
[0267] Unless specifically stated as "unsubstituted," references to
chemical moieties herein are understood to include substituted
variants. For example, reference to an "aryl" group or moiety
implicitly includes both substituted and unsubstituted
variants.
[0268] The term "sulfate" is art-recognized and refers to the group
--OSO.sub.3H, or a pharmaceutically acceptable salt thereof.
[0269] The term "sulfonamide" is art-recognized and refers to the
group represented by the general formulae
##STR00076##
wherein R.sup.7 and R.sup.8 independently represents hydrogen or
hydrocarbyl.
[0270] The term "sulfoxide" is art-recognized and refers to the
group S(O)--R.sup.7,
wherein R.sup.7 represents a hydrocarbyl.
[0271] The term "sulfonate" is art-recognized and refers to the
group SO.sub.3H, or a pharmaceutically acceptable salt thereof.
[0272] The term "sulfone" is art-recognized and refers to the group
--S(O).sub.2--R.sup.7,
wherein R.sup.7 represents a hydrocarbyl.
[0273] The term "thioalkyl", as used herein, refers to an alkyl
group substituted with a thiol group.
[0274] The term "thioester", as used herein, refers to a group
--C(O)SR.sup.7 or --SC(O)R.sup.7 wherein R.sup.7 represents a
hydrocarbyl.
[0275] The term "thioether", as used herein, is equivalent to an
ether, wherein the oxygen is replaced with a sulfur.
[0276] The term "urea" is art-recognized and may be represented by
the general formula
##STR00077##
wherein R.sup.7 and R.sup.8 independently represent hydrogen or a
hydrocarbyl.
[0277] The term "substantially optically pure" or "enantiomerically
pure" means having at least about 95% of the described enantiomer
with no single impurity greater than about 1% and preferably, at
least about 97.5% enantiomeric excess.
[0278] The pharmaceutically acceptable salts of the compounds of
the present invention include the acid addition and the base salts
thereof. A pharmaceutically acceptable salt of a compound of the
present invention may be readily prepared by mixing together
solutions of a compound of the present invention and the desired
acid or base, as appropriate. The salt may precipitate from
solution and be collected by filtration or may be recovered by
evaporation of the solvent. Suitable acid addition salts are formed
from acids which form non-toxic salts and examples are the
hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate,
nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate,
lactate, tartrate, citrate, gluconate, succinate, saccharate,
benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate,
p-toluenesuiphonate and pamoate salts. Suitable base salts are
formed from bases which form non-toxic salts and examples are the
sodium, potassium, aluminium, calcium, magnesium, zinc and
diethanolamine salts. For a review on suitable salts see Berge et
al, J. Pharm. Sci., 66, 1-19, 1977.
[0279] Esters of the compounds of the formulas of the present
invention that are hydrolyzable in vivo include carboxy group(s)
which is/are present in the form of readily hydrolyzable ester
groups. As used herein, esters that are hydrolyzable in vivo
include the lower alkanoyloxy-alkyl esters (e.g., the
acetoxymethyl, pivaloyloxymethyl, 1-acetoxyethyl and
1-pivaloyloxyethyl ester); the lower alkoxycarbonyloxyalkyl esters
(e.g., the methoxycarbonyloxymethyl, 1-ethoxycarbonyloxyethyl and
1-isopropoxycarbonyloxyethyl ester); the lactonyl esters (e.g., the
phthalidyl and thiophthalidyl ester); the lower alkoxymethyl esters
(e.g., the methoxymethyl ester); the lower alkanoylaminomethyl
esters (e.g., the acetamidomethyl ester); the benzyl and
cyanomethyl esters; 2,2-dimethyl-1-oxopropoxy)methyl ester;
2-[(2-methylpropoxy)carbonyl]-2-pentenyl ester;
1-[[(1-methylethoxy)-carbonyl]oxy]ethyl ester; 1-(acetyloxy)ethyl
ester; (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl ester;
1-[[(cyclohexyloxy)carbonyl]oxy]ethyl ester; and
3,3-dimethyl-2-oxobutyl ester. It will be appreciated by those of
ordinary skill in the art that the readily hydrolyzable esters of
the compounds of the present invention can be formed at a free
carboxy group.
[0280] In another embodiment, the present invention is a
pharmaceutical composition that includes a pharmaceutically
acceptable carrier and a RAS-selective lethal compound according to
the present invention, including compounds of formula I, Ia, and/or
Ib, such as for example, compounds 1 and/or 2.
[0281] Another embodiment of the present invention is a method of
treating a condition in a mammal. This method includes the step of
administering to the mammal a therapeutically effective amount of a
RAS-selective lethal compound according to the present invention,
including compounds of formula I, Ia, and/or Ib, such as for
example, compounds 1 and/or 2.
[0282] In the present invention, a "therapeutically effective
amount" is an amount sufficient to effect beneficial or desired
results. In terms of treatment of a mammal, a "therapeutically
effective amount" of a compound is an amount sufficient to treat,
manage, palliate, ameliorate, or stabilize a condition, such as
cancer, in the mammal. A therapeutically effective amount can be
administered in one or more doses.
[0283] The therapeutically effective amount is generally determined
by a physician on a case-by-case basis and is within the skill of
one in the art. Several factors are typically taken into account
when determining an appropriate dosage. These factors include age,
sex and weight of the patient, the condition being treated, the
severity of the condition and the form of the drug being
administered.
[0284] Effective dosage forms, modes of administration, and dosage
amounts may be determined empirically, and making such
determinations is within the skill of the art. It is understood by
those skilled in the art that the dosage amount will vary with the
route of administration, the rate of excretion, the duration of the
treatment, the identity of any other drugs being administered, the
age, size, and species of animal, and like factors well known in
the arts of medicine and veterinary medicine. In general, a
suitable dose of a compound according to the invention will be that
amount of the compound, which is the lowest dose effective to
produce the desired effect. The effective dose of a compound maybe
administered as two, three, four, five, six or more sub-doses,
administered separately at appropriate intervals throughout the
day.
[0285] A compound of the present invention may be administered in
any desired and effective manner: as pharmaceutical compositions
for oral ingestion, or for parenteral or other administration in
any appropriate manner such as intraperitoneal, subcutaneous,
topical, intradermal, inhalation, intrapulmonary, rectal, vaginal,
sublingual, intramuscular, intravenous, intraarterial, intrathecal,
or intralymphatic. Further, a compound of the present invention may
be administered in conjunction with other treatments. A compound or
composition of the present invention maybe encapsulated or
otherwise protected against gastric or other secretions, if
desired.
[0286] While it is possible for a compound of the invention to be
administered alone, it is preferable to administer the compound as
a pharmaceutical formulation (composition). The pharmaceutically
acceptable compositions of the invention comprise one or more
compounds as an active ingredient in admixture with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
other compounds, drugs, ingredients and/or materials. Regardless of
the route of administration selected, the compounds of the present
invention are formulated into pharmaceutically-acceptable dosage
forms by conventional methods known to those of skill in the art.
See, e.g., Remington's Pharmaceutical Sciences (Mack Publishing
Co., Easton, Pa.).
[0287] In one aspect of this embodiment, one or more compounds or
compositions of the present invention may be conjointly
administered to the mammal with an agent that kills cells through
an apoptotic mechanism. In this aspect, the agent is, e.g., a
chemotherapeutic agent--including combinations of chemotherapeutic
agents. Agents useful in the present invention include, e.g., an
EGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin,
carboplatin, cimetidine, caminomycin, mechlorethamine
hydrochloride, pentamethylmelamine, thiotepa, teniposide,
cyclophosphamide, chlorambucil, demethoxyhypocrellin A, melphalan,
ifosfamide, trofosfamide, Treosulfan, podophyllotoxin or
podophyllotoxin derivatives, etoposide phosphate, teniposide,
etoposide, leurosidine, leurosine, vindesine, 9-aminocamptothecin,
camptoirinotecan, crisnatol, megestrol, methopterin, mitomycin C,
ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU),
lovastatin, 1-methyl-4-phenylpyridinium ion, semustine,
staurosporine, streptozocin, phthalocyanine, dacarbazine,
aminopterin, methotrexate, trimetrexate, thioguanine,
mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine
(ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine,
doxorubicin hydrochloride, leucovorin, mycophenolic acid,
daunorubicin, deferoxamine, floxuridine, doxifluridine,
raltitrexed, idarubicin, epirubican, pirarubican, zorubicin,
mitoxantrone, bleomycin sulfate, actinomycin D, safracins,
saframycins, quinocarcins, discodermolides, vincristine,
vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel,
tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR,
estramustine, estramustine phosphate sodium, flutamide,
bicalutamide, buserelin, leuprolide, pteridines, enediynes,
levamisole, aflacon, interferon, interleukins, aldesleukin,
filgrastim, sargramostim, rituximab, BCG, tretinoin, betamethosone,
gemcitabine hydrochloride, verapamil, VP-16, altretamine,
thapsigargin, oxaliplatin, iproplatin, tetraplatin, lobaplatin,
DCP, PLD-147, JM118, JM216, JM335, satraplatin, docetaxel,
deoxygenated paclitaxel, TL-139, 5'-nor-anhydrovinblastine
(hereinafter: 5'-nor-vinblastine), camptothecin, irinotecan
(Camptosar, CPT-11), topotecan (Hycamptin), BAY 38-3441,
9-nitrocamptothecin (Orethecin, rubitecan), exatecan (DX-8951),
lurtotecan (GI-147211C), gimatecan, homocamptothecins diflomotecan
(BN-80915) and 9-aminocamptothecin (IDEC-13'), SN-38, ST1481,
karanitecin (BNP1350), indolocarbazoles (e.g., NB-506),
protoberberines, intoplicines, idenoisoquinolones,
benzo-phenazines, NB-506, and combinations thereof.
[0288] Pharmaceutically acceptable carriers are well known in the
art (see, e.g., Remington's Pharmaceutical Sciences (Mack
Publishing Co., Easton, Pa.) and The National Formulary (American
Pharmaceutical Association, Washington, D.C.)) and include sugars
(e.g., lactose, sucrose, mannitol, and sorbitol), starches,
cellulose preparations, calcium phosphates (e.g., dicalcium
phosphate, tricalcium phosphate and calcium hydrogen phosphate),
sodium citrate, water, aqueous solutions (e.g., saline, sodium
chloride injection, Ringer's injection, dextrose injection,
dextrose and sodium chloride injection, lactated Ringer's
injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and
benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and
polyethylene glycol), organic esters (e.g., ethyl oleate and
tryglycerides), biodegradable polymers (e.g.,
polylactide-polyglycolide, poly(orthoesters), and
poly(anhydrides)), elastomeric matrices, liposomes, microspheres,
oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and
groundnut), cocoa butter, waxes (e.g., suppository waxes),
paraffins, silicones, talc, silicylate, etc. Each pharmaceutically
acceptable carrier used in a pharmaceutical composition of the
invention must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not injurious to
the subject. Carriers suitable for a selected dosage form and
intended route of administration are well known in the art, and
acceptable carriers for a chosen dosage form and method of
administration can be determined using ordinary skill in the
art.
[0289] The pharmaceutical compositions of the invention may,
optionally, contain additional ingredients and/or materials
commonly used in pharmaceutical compositions. These ingredients and
materials are well known in the art and include (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid; (2) binders, such as carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl
cellulose, sucrose and acacia; (3) humectants, such as glycerol;
(4) disintegrating agents, such as agar-agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, sodium
starch glycolate, cross-linked sodium carboxymethyl cellulose and
sodium carbonate; (5) solution retarding agents, such as paraffin;
(6) absorption accelerators, such as quaternary ammonium compounds;
(7) wetting agents, such as cetyl alcohol and glycerol monosterate;
(8) absorbents, such as kaolin and bentonite clay; (9) lubricants,
such as talc, calcium stearate, magnesium stearate, solid
polyethylene glycols, and sodium lauryl sulfate; (10) suspending
agents, such as ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering
agents; (12) excipients, such as lactose, milk sugars, polyethylene
glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa
butter, starches, tragacanth, cellulose derivatives, polyethylene
glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc
oxide, aluminum hydroxide, calcium silicates, and polyamide powder;
(13) inert diluents, such as water or other solvents; (14)
preservatives; (15) surface-active agents; (16) dispersing agents;
(17) control-release or absorption-delaying agents, such as
hydroxypropylmethyl cellulose, other polymer matrices,
biodegradable polymers, liposomes, microspheres, aluminum
monosterate, gelatin, and waxes; (18) opacifying agents; (19)
adjuvants; (20) wetting agents; (21) emulsifying and suspending
agents; (22), solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan; (23)
propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane; (24)
antioxidants; (25) agents which render the formulation isotonic
with the blood of the intended recipient, such as sugars and sodium
chloride; (26) thickening agents; (27) coating materials, such as
lecithin; and (28) sweetening, flavoring, coloring, perfuming and
preservative agents. Each such ingredient or material must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the subject.
Ingredients and materials suitable for a selected dosage form and
intended route of administration are well known in the art, and
acceptable ingredients and materials for a chosen dosage form and
method of administration may be determined using ordinary skill in
the art.
[0290] Pharmaceutical compositions suitable for oral administration
may be in the form of capsules, cachets, pills, tablets, powders,
granules, a solution or a suspension in an aqueous or non-aqueous
liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir
or syrup, a pastille, a bolus, an electuary or a paste. These
formulations may be prepared by methods known in the art, e.g., by
means of conventional pan-coating, mixing, granulation or
lyophilization processes.
[0291] Solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like) may be
prepared by mixing the active ingredient(s) with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
fillers, extenders, binders, humectants, disintegrating agents,
solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, and/or coloring agents. Solid compositions
of a similar type maybe employed as fillers in soft and hard-filled
gelatin capsules using a suitable excipient. A tablet may be made
by compression or molding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared using a suitable
binder, lubricant, inert diluent, preservative, disintegrant,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine. The tablets, and other solid dosage
forms, such as dragees, capsules, pills and granules, may
optionally be scored or prepared with coatings and shells, such as
enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as
to provide slow or controlled release of the active ingredient
therein. They may be sterilized by, for example, filtration through
a bacteria-retaining filter. These compositions may also optionally
contain opacifying agents and may be of a composition such that
they release the active ingredient only, or preferentially, in a
certain portion of the gastrointestinal tract, optionally, in a
delayed manner. The active ingredient can also be in
microencapsulated form.
[0292] Liquid dosage forms for oral administration include
pharmaceutically-acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. The liquid dosage forms may
contain suitable inert diluents commonly used in the art. Besides
inert diluents, the oral compositions may also include adjuvants,
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions may contain suspending agents.
[0293] Pharmaceutical compositions for rectal or vaginal
administration may be presented as a suppository, which maybe
prepared by mixing one or more active ingredient(s) with one or
more suitable nonirritating carriers which are solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the rectum or vaginal cavity and release the active
compound. Pharmaceutical compositions which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such
pharmaceutically-acceptable carriers as are known in the art to be
appropriate.
[0294] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, drops and inhalants. The active compound may be
mixed under sterile conditions with a suitable
pharmaceutically-acceptable carrier. The ointments, pastes, creams
and gels may contain excipients. Powders and sprays may contain
excipients and propellants.
[0295] Pharmaceutical compositions suitable for parenteral
administrations comprise one or more compound in combination with
one or more pharmaceutically-acceptable sterile isotonic aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
suitable antioxidants, buffers, solutes which render the
formulation isotonic with the blood of the intended recipient, or
suspending or thickening agents. Proper fluidity can be maintained,
for example, by the use of coating materials, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. These compositions may also contain suitable
adjuvants, such as wetting agents, emulsifying agents and
dispersing agents. It may also be desirable to include isotonic
agents. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents
which delay absorption.
[0296] In some cases, in order to prolong the effect of a drug, it
is desirable to slow its absorption from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension of crystalline or amorphous material having poor
water solubility.
[0297] The rate of absorption of the drug then depends upon its
rate of dissolution which, in turn, may depend upon crystal size
and crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug may be accomplished by dissolving or
suspending the drug in an oil vehicle. Injectable depot forms may
be made by forming microencapsule matrices of the active ingredient
in biodegradable polymers. Depending on the ratio of the active
ingredient to polymer, and the nature of the particular polymer
employed, the rate of active ingredient release can be controlled.
Depot injectable formulations are also prepared by entrapping the
drug in liposomes or microemulsions which are compatible with body
tissue. The injectable materials can be sterilized for example, by
filtration through a bacterial-retaining filter.
[0298] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0299] Preferably, the mammal is a human. Preferably, the condition
is characterized by cells with enhanced RAS signaling activity. In
another preferred embodiment, the condition is cancer. Preferably,
the cancer is selected from the group consisting of leukemia,
non-small cell lung carcinoma, colon cancer, CNS cancer, melanoma,
ovarian cancer, renal cancer, prostate cancer, breast cancer, and
pancreatic cancer.
[0300] In another embodiment, the present invention is a method of
treating a condition in a mammal, as defined above. This method
includes administering to the mammal a therapeutically effective
amount of a composition or pharmaceutical composition that contains
a pharmaceutically acceptable carrier and a compound according to
formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IV, IVa, IVb, IVc, IVd,
and/or V, including compounds 1-37.
[0301] Additional methods of treating a condition in a mammal are
provided, comprising administering to the mammal a therapeutically
effective amount of a compound selected from the group consisting
of compounds of formula VI, compounds of formula VII, compounds of
formula VIII, compounds of formula IX, and combinations
thereof.
[0302] Further methods of treating a condition in a mammal are also
provided. These methods comprise administering to the mammal a
therapeutically effective amount of a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a
therapeutically effective amount of a compound selected from the
group consisting of compounds of formula VI, compounds of formula
VII, compounds of formula VIII, compounds of formula IX, and
combinations thereof.
[0303] Other methods of treating a condition in a mammal are
provided. These methods comprise administering to the mammal a
therapeutically effective amount of a compound according to any one
of formulas X, Xa, Xb, X1, X2, XII, XIII, XIV, XV, stereoisomers of
Compound 27 of formulas A, B, C, and D, stereoisomers of Cyclized
Compound 27 of formulas E, F, G, and H, or a N-oxide, crystalline
form, hydrate, or pharmaceutically acceptable salt thereof.
[0304] Further methods of treating a condition in a mammal are also
provided. These methods comprise administering to the mammal a
therapeutically effective amount of a compound according to any one
of formulas X, Xa, Xb, X1, X2, XII, XIII, XIV, XV, stereoisomers of
Compound 27 of formulas A, B, C, and D, stereoisomers of Cyclized
Compound 27 of formulas E, F, G, and H, or a N-oxide, crystalline
form, hydrate, or pharmaceutically acceptable salt thereof.
[0305] In these embodiments, preferably, the mammal is a human.
Preferably, the condition is characterized by cells with enhanced
RAS signaling activity. In another preferred embodiment, the
condition is cancer. Preferably, the cancer is selected from the
group consisting of leukemia, non-small cell lung carcinoma, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
prostate cancer, breast cancer, and pancreatic cancer.
[0306] An additional embodiment of the present invention is a
method for identifying RAS-selectively lethal compounds. This
method includes the steps of:
[0307] (a) determining a percent growth inhibition ("% GI") of
candidate compounds in a BJ cell system comprising a plurality of
isogenic cell lines at least one of which is non-tumorigenic and at
least one of which is tumorigenic, wherein a % GI of at least 90%
at a concentration of about 10 .mu.M or less in the candidate
compound in at least one tumorigenic BJ cell line is considered to
be a lethal compound,
[0308] (b) determining a potency profile, in the BJ cell system, of
the candidate compounds from (a) determined to be lethal, wherein
those candidate compounds having at least a 4-fold differential
potency between non-tumorigenic and tumorigenic cell lines in the
BJ cell system are considered to be selective, and
[0309] (c) identifying the selective candidate compounds from (b),
which have a similar potency profile for each of the tumorigenic
cell lines in the BJ cell system, wherein those candidate compounds
having a similar potency profile are considered to be
RAS-selectively lethal compounds.
[0310] Preferably, in this method, the BJ cell system comprises
isogenic cell lines, which are derived from BJ cells. The BJ cell
lines are selected from the group consisting of BJ-TERT,
BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS.sup.V12,
BJ-TERT/LT/RAS.sup.V12/ST, and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12. The construction of an
exemplary BJ cell system is described in more detail below and in,
e.g., Dolma et al., Cancer Cell, 3:285-296 (2003), which is
incorporated by reference as if recited in full herein and in,
e.g., Hahn et al., 1999. In the isogenic cell lines used, oncogenic
elements were sequentially introduced to convert human primary
fibroblast cells into tumorigenic cells. Briefly, BJ cells were
engineered successively to express the catalytic subunit of human
telomerase (hTERT), the SV40 large T and small T antigens (LT and
ST), and an oncogenic allele of HRAS (HRAS.sup.G12v). These cells
lines are identified as BJ-TERT, BJ-TERT/LT/ST, and
BJ-TERT/LT/ST/RAS.sup.V12. Only the BJ-TERT/LT/ST/RAS.sup.V12 cells
form tumors in nude mice. Also, phenotypic changes associated with
expression of each genetic element are indicated along with cell
doubling time. Both RAS.sup.V12 harboring cells
(BJ-TERT/LT/ST/RAS.sup.V12 and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 cells) can form tumors
in nude mice but differ in cell doubling time, which allows
filtering out proliferation-rate dependent lethal compounds.
Preferably, in the method for identifying RAS-selectively lethal
compounds, step (a) is carried out using BJ-TERT/LT/ST/RAS.sup.V12
cells.
[0311] Preferably, in this method, the tumorigenic cell lines in
the BJ cell system are selected from BJ-TERT/LT/ST/RAS.sup.V12,
BJ-TERT/LT/RAS.sup.V12/ST, and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12.
[0312] In another embodiment of the present invention, a method for
characterizing the mechanism of action of a RAS-selectively lethal
candidate compound is provided. In this method, a compound already
determined to be RAS-selectively lethal using the procedures
described above is used in a death kinetics assay with a
tumorigenic BJ cell line using a plasma membrane integrity assay.
Preferably, in the death kinetics assay the tumorigenic BJ cell
line is BJ-TERT/LT/ST/RAS.sup.V12. Preferably, the plasma membrane
integrity assay is a trypan blue assay, which is described in
detail in the Examples. The cell killing effects of hit compounds
are confirmed using the trypan blue assay.
[0313] In this method, if the plasma membrane of the tumorigenic
cell line becomes permeable within about 8-hours of treatment with
a candidate compound, the candidate compound is considered to have
a mechanism of action similar to the compounds of Formula IV. More
particularly, if the plasma membrane of the tumorigenic cell line
becomes permeable within about 8-hours of treatment with a
candidate compound, the candidate compound is considered to have a
mechanism of action similar to compound 27.
[0314] If, however, the plasma membrane of the tumorigenic cell
line becomes permeable within about 12-hours of treatment with a
candidate compound, the candidate compound is considered to have a
mechanism of action similar to the compounds of Formula II. More
particularly, if the plasma membrane of the tumorigenic cell line
becomes permeable within about 12-hours of treatment with a
candidate compound, the candidate compound is considered to have a
mechanism of action similar to compound 3.
[0315] In another embodiment of the present invention, a further
method for characterizing the mechanism of action of a
RAS-selectively lethal compound is provided. In this method,
compound(s) already determined to be RAS-selectively lethal
candidate compound(s) using the procedures described above is/are
used in a counter-screen with a tumorigenic BJ cell line and a
panel of biologically active compounds with known function. Next,
those biologically active compounds that reverse the lethality of
each candidate compound are determined, wherein the specific
pattern of reversed lethality defines a perturbation profile. If
the perturbation profile of a RAS-selectively lethal compound is
the same as, or similar to, a compound having a previously defined
perturbation profile, the two compounds are considered to have the
same or similar mechanisms of action. A flow chart showing the
screening strategy for identifying genetically selective compounds
and ruling out proliferation-dependent compounds is provided in
FIG. 34.
[0316] In this method, it is preferred that the panel of
biologically active compounds includes the following: GSH (Reduced
glutathione), L-Mimosine, Actinomycin D, Cyclohexamide,
z-VAD(OMe)-fmk, Boc-D(OMe)-fmk, DPQ, BHT, alpha-tocopherol (Vit.
E), ALLN (Calpain inhibitor 1), TLCK, EGTA, L-NAME (HCl),
Gd3+(6H2O), Co++(6H2O), 3-methladenine, methyl pyruvate, NMMA,
Desferrioxamine Mesylate, U0126, SP600125, SB-203580, Wortmannin,
LY294002, SH6, Y27632, HA1077 (2HCl), Kenpaullone, LiCl,
(R)-Roscovitine, PP2, SU6656, H89 (2HCl), KN62, Ro 31-8220, and
Rapamycin.
[0317] More particularly, the panel of biologically active
compounds, their respective functions, and the concentration of
each biologically active compound used in the counter screen are
shown in Table 1:
TABLE-US-00001 TABLE 1 Biologically active compounds used for
screening. Compound Function Final Concentration GSH (Reduced
glutathione) inactivate cisplatin, antioxidant 5 mM L-Mimosine cell
cycle blocker, G1 arrest 350 uM Actinomycin D transcription blocker
16 nm Cyclohexamide translation blocker 1.5 um z-VAD(OMe)-fmk
pan-caspase inhibitor 50 uM Boc-D(OMe)-fmk pan-caspase inhibitor 50
uM DPQ PARP inhibitor 10 uM BHT anti-oxidant 400 uM
alpha-tocopherol (Vit. E) anti-oxidant 100 uM ALLN (Calpain
inhibitor 1) cysteine protease inhibitor 45 uM TLCK serine protease
inhibitor 100 uM EGTA calcium chelator 2 mM L-NAME (HCl)
anti-oxidant 300 uM Gd3+(6H2O) direct blocker of I(OGD) current and
656 uM Ca2+ Co++ (6H2O) calcium channel blocker 656 uM
3-methladenine macroautophagy inhibitor 5 mM methylpyruvate
cell-permeant bloenergetic substrate 5 mM NMMA inhibits NO
formation from Arg 250 uM Desferrioxamine Mesylate Fe mobilizer
(inhibits fenton 150 uM chemistry) U0126 MEK1/2 inhibitor 10 uM
SP600125 JNK inhibitor 10 uM SB-203580 SAPK inhibitor 10 uM
Wortmannin PI3K inhibitor 1 uM LY294002 PI3K inhibitor 50 uM SH6
inhibit phosphorylation of Akt 20 uM Y27632 ROCK, PRK2 inhibitor 20
uM HA1077 (2HCl) ROCK, PRK2 inhibitor 50 uM Kenpaullone GSK-3, CDKs
inhibitor 10 uM LiCl GSK-3 inhibitor 10 uM (R)-Roscovitine CDKs
inhibitor 50 uM PP2 Src, Fyn, Lck inhibitor 5 uM SU6656 Src, Fyn,
Lck inhibitor 50 uM H89 (2HCl) inhibit PKA, MSKs, but not RSKs 25
uM KN62 CaM-Ks inhibitor 10 uM Ro 31-8220 inhibit conventional PKCs
5 uM Rapamycin mTOR inhibitor 0.1 uM
[0318] In the present invention, those RAS-selectively lethal
compound(s) that have perturbation profiles in which drug
candidate-induced cell death is inhibited by a protein synthesis
inhibitor, a MEK inhibitor, a general Src-kinase inhibitor, an iron
chelator, and an antioxidant are considered to have the same or
similar mechanism of action as a compound of Formula II, such as
for example, compound 3.
[0319] In the present invention, those RAS-selectively lethal
compound(s) that have perturbation profiles in which drug
candidate-induced cell death is inhibited by reduced glutathione
only are considered to have the same or similar mechanism of action
as a compound of Formula III, such as for example, compound 6.
[0320] In the present invention, those RAS-selectively lethal
compound(s) that have perturbation profiles in which drug
candidate-induced cell death is inhibited by a protein synthesis
inhibitor and an iron chelator are considered to have the same or
similar mechanism of action as a compound of Formula V, such as for
example, compound 36.
[0321] In the present invention, those RAS-selectively lethal
compound(s) that have perturbation profiles in which drug
candidate-induced cell death is inhibited by a MEK inhibitor, an
iron chelator, and a general Src-kinase inhibitor are considered to
have the same or similar mechanism of action as a compound of
Formula IV, such as for example, compound 27, or for example, 1S,3R
Compound 27 or 1S,3R Cyclized Compound 27.
[0322] In another embodiment of the present invention, a further
method for characterizing the mechanism of action of a
RAS-selectively lethal compound is provided. In this method, an
NCI60 test is used to determine a sensitivity profile for each
compound already determined to be a RAS-selectively lethal
candidate compound using the methods described above. A candidate
compound having the same or similar sensitivity profile in the
NCI60 test as a compound according to Formulae I-V is considered to
have the same mechanism of action as such a compound of Formulae
I-V. The NCI60 test is described in more detail in the
Examples.
[0323] Preferably, the methods described above for identifying
RAS-selectively lethal compounds are high-throughput screening
methods. In the present invention, the RAS-selectively lethal
compounds may be selected from any entity capable of functioning as
such. Thus, in the present invention "RAS-selectively lethal
compounds" are those entities that selectively kill cells that
encode or express a mutant RAS protein as described in detail
herein. For example, the RAS-selectively lethal compounds may be
selected from antibodies, RNAi, siRNA, shRNA, antisense sequences,
and small molecules. Preferably, the RAS-selectively lethal
compounds are small molecules.
[0324] The following examples are provided to further illustrate
the compositions and methods of the present invention. These
examples are illustrative only and are not intended to limit the
scope of the invention in any way.
EXAMPLES
Example 1
Materials and Methods
Cell Lines
[0325] BJ-fibroblast-derived cell lines were grown in a 4:1 mixture
of DMEM to M199 supplemented with penicillin and streptomycin
(pen/strep), and 15% heat-inactivated fetal bovine serum (BJ
medium). TRE-fibroblast-derived cell lines and the human lung
carcinoma cell line A549 were grown in DMEM supplemented with
pen/strep and 10% calf serum. MS1 and SVR cells were grown in DMEM
supplemented with pen/strep and 5% calf serum. The human lung
carcinoma cell line Calu-1 and colon carcinoma cell line HCT116
were grown in McCoy's 5A medium supplemented with pen/strep and 10%
calf serum. The human pancreas carcinoma cell line MIA PaCa-2 was
maintained in DMEM supplemented with pen/strep, 10% calf serum, and
2.5% horse serum. The human fibrosarcoma cell line HT1080 was
maintained in DMEM supplemented with non-essential amino acids,
pen/strep, and 10% calf serum.
[0326] The BJ cell system consists of 5 isogenic cell lines that
are derived from common human fibroblast cells. It contains two
non-tumorigenic versions, BJ-TERT and BJ-TERT/LT/ST, and three
tumorigenic versions, BJ-TERT/LT/ST/RAS.sup.V12,
BJ-TERT/LT/RAS.sup.V12/ST, and
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12. The true
oncogenic-RAS-signal dependent lethal compound should have the same
potency in the three tumorigenic cell lines because the three have
oncogenic version of HRAS (RAS.sup.G12V) in common and they have
the same cell-signal context despite being engineered in a
different manner.
[0327] An aim of screening was to identify compounds lethal to
BJ-TERT/LT/ST/RAS.sup.V12 cells, while having minimal effects on
their isogenic cell precursors.
[0328] A549, Calu-1, HCT116 and MIA PaCa-2 cell lines have an
oncogenic mutation in the KRAS gene. Compounds which tested
positive in the oncogenic HRAS primary screen were also tested for
their selectivity against oncogenic KRAS which is more commonly
mutated in human cancers.
Compound Libraries
[0329] Compound libraries were composed of a combination of
synthetic and natural product-like compounds. A number of filters
were applied in choosing these compounds. The TIC library was
selected in part for natural product-like features, including a
number of chiral centers, number of unsaturated rings, and lack of
steroids, which are abundant in natural-product collections. The
BBB library was filtered to eliminate reactive or toxic groups that
are unlikely to make effective drug molecules. The filters included
the presence of chiral centers, the presence of unsaturated rings,
the lack of steroids, and the lack of reactive or toxic groups. In
total, 47,725 small molecules were obtained from different
providers: 2,056 from TimTec; 8,669 from IBS; 17,520 from
Chembridge; 12,240 from Asinex; 5,240 from Life Chemicals; and
2,000 from MicroSource Discovery. All compounds were prepared as 4
mg/mL solutions in DMSO in 384-well polypropylene plates (Greiner,
cat.#781280) and stored at -80.degree. C. These plates are
designated as mother plates. Wells in the outer two rows and
columns were left empty to minimize edge effects from affecting the
screening data.
Identification of Small Molecules that are Synthetic Lethal to
Oncogenic RAS: Primary Screening
[0330] Daughter plates were prepared by diluting mother plates
75-fold in DMEM to obtain a compound concentration of 53.3 .mu.g/mL
with 1.33% DMSO in 384-deep-well polypropylene plates (Greiner,
cat.#781270). Assay plates were prepared by seeding 1,000
BJ-TERT/LT/ST/RAS.sup.V12 cells per well in 36 .mu.L of growth
media in black, clear-bottom, 384-well plates (Corning Inc., cat.
#3712). Columns 3-22 and rows C through N were treated with
compounds from a daughter library plate by transferring 4 .mu.L
from the daughter library plate to the assay plates. The final
compound concentration in the assay plates was 5 .mu.g/mL with 0.1%
DMSO. All liquid handling was carried out using a Biomek FX AP384
module (Beckman Coulter). The assay plates were incubated for 48
hours at 37.degree. C. in a humidified incubator containing 5%
CO.sub.2. Cell viability was measured using alamar blue
(Invitrogen, cat.# DAL1100), which detects changes in cellular
reductive potential (Nociari et al., 1998). Subsequently, percent
growth inhibition was calculated. All experiments were performed in
triplicate and median percent growth inhibition was determined.
Compounds that inhibited alamar blue metabolism in
BJ-TERT/LT/ST/RAS.sup.V12 cells more than 50% were considered
initial hits.
Retesting Compounds in Dilution Series in Three BJ Cell Lines
[0331] Empty "step-daughter plates" were filled with 50 .mu.L DMEM,
except for columns 3 and 13, where 100 .mu.L of hit compound
solution were cherry-picked from daughter plates. After compound
transfer, 2-fold dilution series across columns 3 through 12 and
columns 13 through 22 was made by transferring 50 .mu.L of compound
solution to the next column successively (10-point dilution
series), with extensive mixing. Hit-picking and 2-fold dilution
series were carried out using Biomek FX Span-8 module (Beckman
Coulter). Assay plates were prepared by seeding 1,000 BJ-TERT,
BJ-TERT/LT/ST, and BJ-TERT/LT/ST/RAS.sup.V12 cells per well in 36
.mu.L growth medium. Cells in assay plates were treated with
compounds from step-daughter plates by transferring 4 .mu.L. The
final concentration of the compound was 5.33 .mu.g/mL to 0.01
.mu.g/mL in this 10-point, 2-fold dilution series. Assay plates
were returned to the culture incubator and maintained for 48 hours
before adding alamar blue.
Retesting Compounds in Dilution Series in Four BJ Cell Lines
[0332] Fresh daughter plates were made from the mother plate and
cherry-picked hits from the first round, 2-fold dilution series
experiment as described above. Assay plates were prepared by
seeding 1,000 BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS.sup.V12 or
BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells per well in
32 .mu.L growth media. Cells in the assay plates were treated with
compounds from the step-daughter plate by transferring 8 .mu.L
solution. The final concentration of the compound was 10.66
.mu.g/mL to 0.02 .mu.g/mL in a 10-point, 2-fold dilution series
manner. Assay plates were returned to the culture incubator and
maintained for 48 hours before adding alamar blue.
Alamar Blue Assay
[0333] Cells were seeded in 384-well assay plates, treated with
candidate compounds, and incubated in the culture incubator in 40
.mu.L culture volume per well. After 24 or 48 hours of compound
treatment, 10 .mu.L of 50% alamar blue solution in growth medium
was transferred to the assay plates, which resulted in 10% final
concentration alamar blue. Plates were incubated further for 16
hours to allow reduction of alamar blue, which results in the
generation of red fluorescence. The fluorescence intensity was
determined using a Victor 3 plate reader (Perkin Elmer) with a 535
nm excitation filter and a 590 nm emission filter.
Vi-Cell Analysis (Trypan Blue Assay)
[0334] 200,000 cells were seeded in 6-well plates and treated with
the indicated amount of 2-fold dilution series concentration of a
candidate compound in 2 mL BJ growth medium. After 24 hours, cells
were released with trypsin/EDTA, harvested in 1.1 mL growth medium,
and transferred to a Vi-Cell (Beckman Coulter) disposable cup.
After, trypan blue staining, 100 images of sample were taken and
analysis of the images was carried out automatically by a Vi-Cell
cell viability analyzer (Beckman Coulter).
Counter Screening with Biologically Active Compounds
[0335] Biologically active compounds listed in Table 1 were
dissolved in BJ medium at 10.times. final concentration and
aliquoted into eppendorf tubes. 100 .mu.L of each 10.times.
solution was transferred to column 1 to column 12 or column 13 to
column 24 of a single row in a 384-well daughter plate. This plate
was designated as `10.times. biologically active plate`. In order
to prepare lethal compound solution, Erastin and compounds 3, 6,
27, and 36 were dissolved in BJ medium at a concentration of 100
.mu.g/mL, 400 .mu.g/mL, 400 .mu.g/mL, 100 .mu.g/mL, and 200
.mu.g/mL, respectively. Empty daughter plates were filled with 50
.mu.L BJ media except for columns 5 and 13 where 100 .mu.L of each
lethal solution was transferred. After lethal solution transfer,
2-fold dilution series across columns 5 to 12 and columns 13 to 20
was made by transferring 50 .mu.L compound solution to the next
column successively (8-point dilution series), with mixing. These
five plates were each designated `10.times. lethal plate`. Five
assay plates were prepared by seeding 4,000
BJ-TERT/LT/ST/RAS.sup.V12 cells per well in 32 .mu.L of growth
media in black, clear bottom 384-well plates. Cells in assay plates
were first treated with biologically active compounds by
transferring 4 .mu.L from the 10.times. inhibitor plate. Cells were
then treated with each lethal compound in a 2-fold dilution series
by transferring 4 .mu.L solution from each 10.times. lethal plate.
Assay plates were returned to the culture incubator and maintained
for 24 hours before adding alamar blue. Percent growth inhibition
(% GI) was calculated by the following formula using fluorescence
intensity values:
% GI=100*(1-(X-N)/(P-N))
wherein X is the fluorescent intensity value measured at 590 nm for
cells treated with biologically active compound and lethal
compound; N is the fluorescent intensity value measured at 590 nm
for a control containing no cells (i.e. biologically active
compound in growth media); and P is the fluorescent intensity value
measured at 590 nm for a control containing no lethal compound
(i.e., cells treated with biologically active compound). Counter
Screening with Iron Chelators
[0336] BJ-TERT/LT/ST/RAS.sup.V12 cells were seeded in 384-well
assay plates at a concentration of 4,000 cells per well in 32 .mu.L
volume. Cells were treated with iron chelators by transferring 4
.mu.L of 1 mM deferoxamine mesylate (Calbiochem, cat.#252750), 500
.mu.M compound 1a (Chembridge, cat.#5175092), 1 mM compound 2 (Ryan
Scientific, cat.#LT01784723), or 100 .mu.M compound 311
(Chembridge, cat.#5135701) in growth media to the assay plate.
Then, 4 .mu.L was transferred from the `10.times. lethal plate`
(described in `counter screening with biologically active
compounds`) to the assay plate in order to induce cell death. Assay
plates were incubated for 24 hours and alamar blue was added to the
plates at a final concentration of 10%.
Effect of Cell Cycle Inhibitors on Compound 36-Induced Cell
Death
[0337] BJ-TERT/LT/ST/RAS.sup.V12 cells were seeded in a 384-well
assay plate at a concentration of 4,000 cells per well in 32 .mu.L
culture volume. Cells were treated with cell cycle inhibitors by
transferring 4 .mu.L of 500 .mu.M apigenin (Sigma, cat.#A3145), 1.5
mM monastrol (Sigma, cat.#M8515), 1 mM olomoucine (Sigma,
cat.#O0886), 2 .mu.M Nocodazole (Sigma, cat.#M1404), 6 mM
Hydroxy-urea (Sigma, cat.#H8627), 0.1 .mu.M colchicine (Sigma,
cat.#D6165), or 50 .mu.M Rapamycine (Sigma, cat#RO395) in growth
media to the assay plate. Then, 4 .mu.L was transferred from a
daughter plate that had an 8-point, 2-fold dilution series of
Compound 36 (starting at column 5, 200 .mu.g/mL and ending at
column 12, 1.5625 .mu.g/mL in growth media) to the assay plate. The
assay plate was incubated for 24 hours and alamar blue was added to
the assay plate at a final concentration of 10%.
Monitoring Cellular Iron Level Using Flow Cytometry
[0338] Cells were seeded in 6-well plates (200,000 cells per well)
in BJ medium. The next day, the cell monolayer was washed with
phosphate buffered saline ("PBS") twice and stained with 5 .mu.M of
Phen Green SK, diacetate (Molecular Probe, cat.#P14313) in PBS by
incubating the plate for 15 minutes in a culture incubator. Cells
were released with trypsin/EDTA, harvested in 2 mL PBS, and
centrifuged at 1,000 rpm for 5 min. The cell pellet was
re-suspended in 1 mL of PBS, the cell suspension was transferred to
disposable FACS tubes, and the fluorescence profile of the sample
was monitored using a FACSCalibur system (BD Biosciences).
Western Blot
[0339] Monitoring cleavage of PARP and caspase-3 upon compound
treatment
[0340] 2.times.10.sup.6 BJ-TERT/LT/ST/RAS.sup.V12 cells were seeded
in 10 cm dishes and treated with 1 .mu.M staurosporine, 10 .mu.g/mL
erastin, 20 .mu.g/mL Compound 3, and 1 .mu.g/mL Compound 27 for 16
h. Both dying cells and live cells in each 10 cm dish were
harvested and collected in the same 15 mL tubes by centrifuging
cell suspension at 1,000 rpm for 5 min. Cell pellets were washed
three times with PBS and cells were lysed in 200 .mu.L of
denaturing lysis buffer (50 mM HEPES KOH [pH 7.4], 40 mM NaCl, 2 mM
EDTA, 1.5 mM Na.sub.3VO.sub.4, 50 mM NaF, 10 mM sodium
pyrophosphate, 10 mM sodium .beta.-glycerophosphate, 0.5% Triton
X-100, and protease inhibitor tablet [Roche, cat.#11836170001]).
Protein content was quantified using a Bio-Rad protein assay
reagent (Bio-Rad, cat.#500-00006). Equal amounts of protein were
resolved on SDS-polyacrylamide gels. The electrophoresed proteins
were transblotted onto a PVDF membrane, blocked with 5% milk, and
incubated with rabbit primary antibodies specific to: PARP (Santa
Cruz, cat.#sc-7150), cleaved caspase-3 (Cell Signaling Technology,
cat.#9661) overnight at 4.degree. C. The membrane was then
incubated in IRDye 800 goat anti-rabbit antibody (Li-cor Bioscience
cat.#926-32211) at 1:3,000 dilutions for 45 min at room
temperature. After washing off the unbound antibodies, membranes
were scanned using the Odyssey Imaging System (Li-cor
Bioscience).
Basal Expression Level of TfR1
[0341] BJ-TERT, BJ-TERT/LT/ST, and BJ-TERT/LT/ST/RAS.sup.V12 cells
were seeded prior to the experiment in 10-cm dishes and allowed to
grow to 80% confluence. The cell monolayer was washed three times
with PBS and cells were lysed in 200 .mu.L of denaturing lysis
buffer (50 mM HEPES KOH (pH 7.4), 40 mM NaCl, 2 mM EDTA, 1.5 nN
Na.sub.3VO.sub.4, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM
sodium .beta.-glycerophosphate, 0.5% Triton X-100, and protease
inhibitor tablet (Roche, cat.#11836170001)). Protein content was
quantified using a Bio-Rad protein assay reagent (Bio-Rad,
cat.#500-00006) according to the manufacturer's instructions. Equal
amounts of protein were resolved on SDS-polyacrylamide gels. The
electrophoresed proteins were transblotted onto a PVDF membrane,
blocked with 5% milk, and incubated with goat primary antibodies
specific to transferrin receptor 1 (Santa Cruz, cat.#sc-7087)
overnight at 4.degree. C. The membrane was then incubated in IRDye
800 rabbit anti-goat antibody (Rockland Immunochemicals
cat.#605-432-013) at 1:3,000 dilution for 45 minutes at room
temperature. After washing off the unbound antibodies, membranes
were scanned using the Licor Odyssey.TM. Imaging System.
TfR1 Expression Level Upon Compound Treatment
[0342] 2.times.10.sup.6 BJ-TERT/LT/ST/RAS.sup.V12 cells were seeded
in 10 cm dishes and treated with the iron chelators deferoxamine
mesylate, 2, or 311, or Erastin, Compound 3, Compound 6, Compound
27, or Compound 36 at the indicated concentrations for 24 hours.
Both dying cells and live cells in each 10 cm dish were then
harvested and collected in the same 15 mL tubes by the centrifuging
cell suspension at 1,000 rpm for 5 minutes. Cell pellets were
washed three times with PBS and cells were lysed in 200 .mu.L of
denaturing lysis buffer. Proteins in the cell lysate were
quantified and resolved on SDS-polyacrylamide gels as described
above. The membrane was incubated with anti-transferrin-receptor=1
(Santa Cruz, cat.#sc-7087) and then with IRDye 800 rabbit anti-goat
antibody (Rockland Immunochemicals, cat.#605-432-013).
shRNA Knock Down Experiments
Virus Production
[0343] Lentiviral plasmids encoding shRNA targeting VDAC2
(cat.#SHGLY-NM.sub.--003375), VDAC3 (cat.#SHGLY-NM.sub.--005662),
HRAS (cat.#SHGLY-NM.sub.--005343), NRAS
(cat.#SHGLY-NM.sub.--002524), KRAS (cat.#SHGLY-NM.sub.--004985 and
SHGLY-NM.sub.--033360), or TfR1 (Sigma,
cat.#SHGLY-NM.sub.--003234). All shRNA clones were purchased from
Sigma's MISSION.RTM. shRNA collection. Plasmid DNA was purified
using a HiSpeed Plasmid Midi kit (Qiagen, cat.#12643) according to
the manufacturer's instructions. On day 1, 2.times.10.sup.6 293T
cells were seeded in 10 cm tissue culture dishes. On day 2, 2.8
.mu.g of the shRNA-plasmid construct and 2.5 .mu.g of pDelta8.9 and
0.28 .mu.g of pVSV-G helper plasmids were co-transfected into the
293T cells using FuGENE.RTM. 6 Transfection Reagent (Roche,
cat.#11-814-443-001). On day 3, the medium was replaced with 7.5 mL
of VCM (Viral Collection Media) that consisted of DMEM supplemented
with penicillin and streptomycin (pen/strep), and 30% Hyclone iFCS
(Hyclone, cat.#83007-198). On day 4, in the morning, the
supernatant containing virus was harvested to empty 50 mL conical
tubes and 7.5 mL of fresh VCM was added back to the virus producing
293T cell monolayer. The VCM was harvested and replaced again in
the evening. On day 5, in the morning, the supernatant was
harvested and the 293T cell culture was bleached. The collected
virus supernatant was filtered through 0.45 .mu.m syringe filter
(Nalgene, cat.#190-9945), aliquoted in 2 mL to the cryovials, and
stored at -80.degree. C. freezer until the time of use.
Generation of Stable Cell Lines
[0344] On day one, 3.times.10.sup.5 Calu-1 cells or
2.times.10.sup.5 HT1080 cells were seeded in 10-cm dishes; on day
2, frozen virus stock was thawed at 37.degree. C., hexadimethrine
bromide (polybrene) (Sigma, cat.#H9268) was added to the virus
solution at a final concentration of 8 .mu.g/mL and growth media
from cell culture was replaced with the virus/polybrene mixture.
The culture was returned to the tissue culture incubator for 2
hours, with rocking of the dishes every 30 min in order to prevent
drying. After 2 h, 10 mL growth medium was added, the culture was
incubated for 2 days, and cells stably expressing shRNA were
selected by adding puromycin (Sigma, cat.#P9620) at a final
concentration of 1.5 .mu.g/mL.
Monitoring Drug Sensitivity
[0345] On the day of experiment, assay plates were prepared by
seeding 3,000 HT1080, HT1080-pLKO, HT1080-TfR1 KD, Calu-1,
Calu-1-pLKO, or Calu-1-509 cells per well in 36 .mu.L of growth
media to black, clear bottom 384-well plates. Also, assay plates
were prepared by seeding 1,500 shRNA infected HT1080 or Calu-1
cells per well. Cells in assay plates were treated with each lethal
compound in a 2-fold dilution series by transferring 4 .mu.L
solution from a 10.times. lethal plate of Erastin, Compound 3,
Compound 27, or Compound 36. Assay plates were returned to the
culture incubator and maintained for 24 hours before adding alamar
blue. Percent growth inhibition (% GI) was calculated using
fluorescence intensity values.
Real-Time RT-PCR
[0346] Total RNA was extracted using RNeasy kit (Qiagen,
cat.#74104) as described in the manufacturer's handbook. One
microgram of RNA sample was subject to reverse transcription
reaction using TaqMan.RTM. Reverse Transcription Reagents (Applied
Biosystems, cat.# N8080234) according to the manufacturer's
instruction. Then, Q-PCR was carried out using Power SYBR.RTM.
Green PCR Master Mix (Applied Biosystems, cat.# 4367659) and 7300
Real-Time PCR System (Applied Biosystems). The sequence of primers
for Q-PCR is shown in Table 11. The mRNA level of human acidic
ribosomal phosphoprotein PO was measured using the SEQ ID NOs. 17
and 18 primers and used as a reference for quantification.
Results
[0347] Compounds that inhibited alamar blue metabolism in
BJ-TERT/LT/ST/RAS.sup.V12 cells more than 50% were considered
initial hits and tested in an extensive two-fold dilution series in
BJ-TERT, BJ-TERT/LT/ST and BJ-TERT/LT/ST/RAS.sup.V12 cells. The TIC
library had a large number of hits (897 compounds). In this case,
another round of primary screening was carried out using
BJ-TERT/LT/ST cells and calculated delta percent growth inhibition
(.DELTA.% GI), as percent growth inhibition (% GI) in
BJ-TERT/LT/ST/RAS.sup.V12 cells minus % GI in BJ-TERT/LT/ST cells.
66 compounds showed up as hits with >50 .DELTA.% GI. Also of
interest were the 224 compounds in the TIC library that showed more
than 90% growth inhibition in BJ-TERT/LT/ST/RAS.sup.V12 cells,
because of their strong growth inhibition activity. In total, 533
compounds (66 TIC .DELTA.% GI hits, 224 TIC>90% GI hits, 197
BBB>50% GI hits, and 46 MS>50% GI hits) were selected from
the primary screening and tested further.
[0348] In the subsequent two-fold dilution series testing,
selectivity was calculated as the ratio of GI.sub.50 values in
BJ-TERT/LT/ST and BJ-TERT/LT/ST/RAS.sup.V12 cells. Hit compounds
showing at least 4-fold selectivity for HRAS.sup.G12V were further
tested in BJ-TERT/p53DD/CDK4/cyclinD1/ST/RAS.sup.V12 (DRD) cells
that were engineered to express hTERT, oncogenic HRAS
(HRAS.sup.G12V), dominant negative p53, and constitutively active
CDK4/cyclinD, which inactivates the RB protein. The
p53DD/CDK4/cyclinD1 combinations substitute for the SV40 large T
oncoprotein (Dolma et al., 2003; Hahn et al., 2002). DRD cells are
also derived from BJ primary cells and the effects of mutations in
both lines should be similar. Accordingly, ideal synthetic lethal
compounds should have similar growth inhibitory potency in both
cell lines. The use of DRD cells as a counter-screen enabled the
elimination of compounds that (i) have idiosyncratic activity in
BJ-TERT/LT/ST/RAS.sup.V12 cells, (ii) are simply more potent in
rapidly dividing cells (DRD cells divide more slowly than
BJ-TERT/LT/ST/RAS.sup.V12 cells), and (iii) target the SV40 viral
protein rather than an endogenous oncogenic signaling pathway.
[0349] Seventy compounds from the two-fold dilution series
experiments were used as `inputs` for the DRD cell line test (i.e.
testing whether these compounds were equally active in DRD and
BJ-TERT/LT/ST/RAS.sup.V12 cells). These 70 compounds included
well-known anti-neoplastic compounds, such as camptothecins,
artemisins, prostaglandins, teniposide, vinblastine, and taxol.
None of these known anti-neoplastic compounds was successful in the
DRD cell test (i.e. they were not equally active in DRD cells and
BJ-TERT/LT/ST/RAS.sup.V12 cells).
[0350] However, seven compounds with unknown functions or
activities were identified (FIG. 1) that passed the DRD cell line
test. These seven compounds were divided into four groups based on
their structures. The most potent, selective, and active hits from
each group were identified. These compounds included both synthetic
compounds and natural product derivatives with four different
scaffolds (FIG. 1). The most potent compound inhibited the growth
of cancer cells at a concentration of a few nanomolar. The most
selective compound showed 16-fold selectivity towards HRAS.sup.G12V
harboring cells. Below, the biological activities of these four hit
compounds and their structural analogs are summarized.
##STR00078##
[0351] Compound 3 is a synthetic compound derived from
3-quinolinecarboxylic acid (FIG. 2A). This compound showed moderate
potency (IC.sub.90=10 .mu.g/mL). The degree of selectivity against
HRAS.sup.G12V was 4-8 folds, as measured by alamar blue reduction
(FIG. 2A). The IC.sub.90 value for Compound 3 in
BJ-TERT/LT/ST/RAS.sup.V12 cells was almost the same as in DRD
cells, which indicates that its activity depends on oncogenic RAS
signaling rather than proliferation rate (FIG. 2A). A trypan blue
exclusion assay was performed to confirm the growth inhibitory
effect and the HRAS.sup.V12 selectivity. This was performed in a
Vi-Cell (Beckman Coulter), which stains cells with trypan blue,
takes 100 images, and analyzes the images to calculate the number
of viable cells (i.e. cells excluding trypan blue). This assay
eliminates false positives caused by auto-fluorescence or redox
activity from a compound in the alamar blue assay. Trypan blue
exclusion assays with Compound 3 revealed a similar level of
potency and selectivity as the alamar blue assay (FIG. 3).
[0352] Structural analogs of Compound 3 with more than 90%
similarity, as assessed by the Tanimoto coefficient, were obtained.
These analogs were tested in the engineered BJ cell system to
define their structure-activity relationship (FIG. 2A and FIG. 4).
Six analogs that have substituents on the bottom phenyl ring other
than fluoro lost growth inhibitory activity, suggesting a critical
role for this fluorophenyl functionality in selective growth
inhibition (FIG. 2A--47324-12 and FIGS. 4--47324-6, -13, -17, -21,
and -32). Two analogs lacking a chloro substituent on the upper
phenyl ring had a similar level of potency in
BJ-TERT/LT/ST/RAS.sup.V12 cells as the original hit, but were
inactive in the DRD cell line, implying that the presence of the
chlorophenyl increases the compound's genuine selectivity for
oncogenic RAS signaling (FIG. 2A-Compound 4 and FIGS. 4--47324-4).
Three analogs differing only in the length of carboxylic acid alkyl
chain showed varied potency and selectivity, which opens the
possibility of modifying this region to obtain better analogs,
especially in terms of solubility, and to create affinity reagents
(FIG. 2A-Compound 5 and FIGS. 4--47324-2 and -8).
[0353] To assess potential mechanisms of action underlying Compound
3-induced cell death, counter screening with 36 compounds having
known biological functions was conducted (FIG. 5). These 36
compounds were selected based on their known property of inhibiting
aspects of cell death signaling or kinase-signaling pathways
related to RAS (FIG. 5). Multiple compounds were able to inhibit
Compound 3-induced cell death, including anti-oxidants (GSH, BHT,
Vit. E), iron chelators (L-mim, DFOM), and a protein synthesis
inhibitor (cycloheximide) (FIG. 5). It is likely that Compound 3
generates iron-mediated reactive oxygen species (ROS) to kill these
tumor cells. In addition, this process requires new protein
synthesis.
[0354] Several kinase inhibitors suppressed Compound 3-induced cell
death, but most importantly, U0126, a MEK1/2 inhibitor, completely
blocked Compound 3-induced cell death, demonstrating that it is
specifically the RAS-RAF-MEK pathway that causes Compound 3
sensitivity, as opposed to other aspects of RAS signaling (FIG.
5).
[0355] To ensure that the oncogenic-RAS-selective-lethal nature of
these compounds was relevant beyond the BJ cell system, Compound 3
was tested in another pair of human fibroblast cell lines, with or
without oncogenic RAS, known as the TRE cell system (Dolma et al.,
2003; Lessnick et al., 2002). TRE cells were derived from primary
human fibroblasts of another patient and were engineered to express
the catalytic subunit of human telomerase (hTERT). The cells were
further engineered to sequentially express human papilloma virus
E6, E7 antigen, SV40 virus ST antigen, and oncogenic HRAS
(HRAS.sup.G12V). Two of the cell lines were named TRE/E6/E7/ST and
TRE/E6/E7/ST/RAS.sup.V12. When tested in TRE, TRE/E6/E7/ST and
TRE/E6/E7/ST/RAS.sup.V12 cells, the selectivity of Compound 3
against oncogenic RAS was manifest, although its growth inhibition
potency was less than in the BJ cell system (FIG. 2B).
[0356] Among the three RAS genes (HRAS, KRAS, NRAS), the BJ cell
system employed HRAS mutations to initiate oncogenic signals. All
three RAS proteins can activate common down stream effectors but,
for unknown reasons, human cancer cells have biased mutation rates
among the three RAS genes; more than 70% contain KRAS mutations,
25% NRAS mutations, and less than 5% HRAS mutations (Rodenhuis,
1992). Since KRAS mutations are most prevalent in human cancers,
the activity of Compound 3 in KRAS-mutation-harboring cancer cells
was tested (FIG. 2B). Compound 3 showed less activity in
KRAS-mutation-harboring cancer cells, raising the possibility that
this compound preferentially targets HRAS signaling. This makes
Compound 3 less attractive as a therapeutic agent for a broad array
of cancers, but potentially useful as a research tool to define
differences in downstream signaling pathways activated by HRAS and
KRAS.
[0357] Overall, Compound 3 has selective lethality toward oncogenic
HRAS signaling, but tends to be cytostatic and less potent in cells
other than BJ-derived cell lines. One advantage of this compound is
that it is relatively easy to modify the original hit compound,
because it is a simple synthetic compound. Synthesis of additional
structural analogs based on this preliminary SAR study may yield
more selective and potent compounds that can be used as research
tools.
##STR00079##
[0358] Compound 6 is a glycosylated echinochrome, a pigment of sea
urchin which has a red color (Nishibori, 1961). Echinochromes are
known to have biological activities, such as free radical
scavenging and iron chelation, but anti-cancer effects have not
been addressed (Lebedev et al., 2005). The
5,8-dihydroxy-1,4-naphthoquinone backbone is also found in
anthracyclines (doxorubicin, daunorubicin) and anthracenediones
(e.g., mitoxantrone). However, none of these traditional
chemotherapeutic agents were active in counter-screening using the
DRD cell line, suggesting some unique properties of Compound 6.
[0359] Compound 6 had reasonable potency in
BJ-TERT/LT/ST/RAS.sup.V12 cells (IC.sub.90=2.5 .mu.g/mL) and the
degree of selectivity towards oncogenic HRAS was 4-8 fold in the BJ
cell system (FIG. 6A). The growth inhibitory potential in DRD cells
was slightly less than that in BJ-TERT/LT/ST/RAS.sup.V12 cells, but
still greater than that in BJ-TERT/LT/ST cells. This suggests that
Compound 6 is an oncogenic-RAS-selective lethal compound and that
the presence of idiosyncratic factors in BJ-TERT/LT/ST/RAS.sup.V12
cells increases their sensitivity further (FIG. 6A). Vi-Cell
analysis of trypan blue exclusion confirmed the potency and
selectivity of Compound 6 (FIG. 7). When this compound was tested
in the TRE cell model, it showed consistent selectivity towards
oncogenic HRAS (FIG. 6B). It also showed reasonable potency in
inhibiting growth of three other mutant-KRAS-harboring cancer cell
lines (FIG. 6B).
[0360] Based on previous reports about echinochrome, it was
expected that reactive oxygen species scavengers or iron chelators
might suppress Compound 6-induced cell death. However, the counter
screening revealed that only reduced glutathione (GSH) was able to
inhibit Compound 6-induced cell death, implying that the mechanism
of action for this compound is different from that of the parental
echinochrome (FIG. 8). GSH can deplete reactive oxygen species
(ROS), but two other ROS scavenger molecules, vitamin E (vit. E)
and butylated hydroxytoluene (BHT), didn't suppress Compound
6-induced cell death. These results imply that ROS do not play a
major role in Compound 6 lethality. It is possible that Compound 6
is conjugated to GSH, which results in the inactivation of the
compound or excretion of the Compound 6-GSH conjugate from the
intracellular environment. This mechanism has been seen with other
anti-cancer drugs (Borst et al., 2000; Kartalou and Essigmann,
2001). A calcium/calmodulin protein kinase inhibitor (CaM-Ks
inhibitor), KN62, also protected BJ-TERT/LT/ST/RAS.sup.V12 cells
from Compound 6-induced death. However, the role of CaM-Ks in the
activity of Compound 6 is not straightforward, because EGTA, a
calcium chelator, slightly increased sensitivity of
BJ-TERT/LT/ST/RAS.sup.V12 cells to Compound 6 (FIG. 8).
[0361] Structural analog searching identified 22 available
compounds that have a 1,4-napthoquinone backbone with various
carbohydrate appendages (FIG. 9). Active analogs typically have one
O-linked carbohydrate and slightly different modifications in the
core 1,4-napthoquinone ring. However, none of them were as
selective as Compound 6, implying the existence of near-optimal
features in this parent compound. The ethyl substituent on the
hydroxyl-containing ring is a unique feature in Compound 6. A
search for structural analogs that have the 1,4-napthoquinone
backbone with a carbon chain of more than 2 carbons in length
resulted in the identification of lapachol, a known compound with
anticancer activity.
[0362] Lapachol is a biologically active constituent of Tabebuia
impetiginosa tree bark that has been used for diminishing fever and
pain in patients in South America. Early in the 20.sup.th century,
lapachol and related analogs were synthesized, and some compounds,
including lapachol and beta-lapachone, showed anti-cancer activity
in vitro and in vivo (da Consolacao et al., 1975; Pardee et al.,
2002). Glycosylated lapachol was tested in the BJ cell model, a
similar degree of potency and selectivity was observed, although
the potency in DRD cells was less than that of Compound 6. (FIG.
9). The National Cancer Institute has tested lapachol in their in
vitro and in vivo cancer models and concluded that although it is
active in tumor growth inhibition, the background cytotoxicity is
too high to warrant clinical use (Block et al., 1974). However, its
cancer-cell-specific action, as well as broad spectrum range of
anticancer activity, attracted many researchers; development of
anti-cancer drugs from lapachol or beta-lapachone is ongoing
(Pardee et al., 2002). Based on the structural analysis, it would
be interesting to synthesize and test analogs of lapachol with a
short aliphatic chain, as is found in Compound 6.
##STR00080##
[0363] Compound 36 is a natural product derivative containing the
labdane diterpene skeleton found in compounds from plant and marine
sources. A number of labdane diterpene derivatives have been
reported to have a wide range of biological activities, including
antibiotic, anti-inflammatory, cardiotonic, and cytotoxic effects
(Singh et al., 1999). However, a cancer-cell-selective growth
inhibitory effect has not been described for this group of
compounds. Compound 36 showed reasonable potency for growth
inhibition of BJ-TERT/LT/ST/RAS.sup.V12 cells. The IC.sub.90 value
was 1 .mu.g/mL (FIG. 10A). The degree of selectivity between
BJ-TERT/LT/ST/RAS.sup.V12 and BJ-TERT was up to 16-fold (FIG. 10A).
BJ-TERT/LT/ST cells showed intermediate sensitivity, implying that
the activity of the compound depends on altered signaling by LT/ST,
while oncogenic RAS signaling further augments sensitivity. Vi-Cell
analysis confirmed the growth inhibitory potency and selectivity
(FIG. 11).
[0364] The sensitivity of DRD cells was close to that of
BJ-TERT/LT/ST cells. However, the increase in sensitivity upon
activation of oncogenic RAS signaling was seen in two additional
pairs of isogenic cell lines with or without oncogenic RAS (TRE
cell lines and MS1/SVR. MS1 and SVR cells are mouse pancreatic
endothelial cells that were engineered to express SV40 LT protein
with (SVR cells) or without HRAS.sup.G12V (MS1 cells) (Arbiser et
al., 1997) (FIG. 10B)). Compound 36 was active in other
KRAS-mutation-harboring cancer cell lines with varying degrees of
sensitivity (FIG. 10B).
[0365] Counter screening with bioactive compounds revealed that
iron chelators (L-Mim and DFOM) and a protein synthesis inhibitor
(cycloheximide) were effective in suppressing Compound 36-induced
cell death (FIG. 12). Since L-mimosine is know to have the activity
of both iron chelation and cell cycle blocking, other cell cycle
blockers were tested in order to dissect the function needed for
suppressing Compound 36-induced cell death. None of cell cycle
blockers were effective in cell death suppression, but other iron
chelators were effective suppressors, indicating that iron
chelation is likely the cause of the protective effect of
L-minosine (FIG. 10C). Compound 36-induced cell death requires new
protein synthesis, based on the effect of cycloheximide.
Selectivity towards HRAS.sup.G12V is mediated by RAS downstream
signaling other than RAS-RAF-MEK pathway, based on the inability of
MEK1/2 inhibitors to suppress Compound 36.
[0366] Structural analog searching resulted in identification of 15
analogs (FIG. 13). One analog differed only in the location of
double bond in the transdecalin ring; this analog had slightly
lower potency and selectivity than the original hit (FIG.
10A-Compound 36). An analog that does not have the thiourea
functionality in the thiosemicarbazone moiety was not active in
inhibiting cell growth in the BJ cell system, implying that this
moiety is critical for activity (FIG. 10A--24578-7). Indeed, an
analog was found that differs only in an oxygen atom substitution
for nitrogen. This analog was not active in the BJ cell system,
confirming the importance of the thiosemicarbazone group (FIG.
10A--24578-3).
[0367] Thiosemicarbazones are known to have multiple functions in
biological systems. One particular class, called heterocyclic
thiosemicarbazones (HCTs), binds iron and induces cytotoxicity in
various cancer cells (Liu et al., 1995). Some HCTs, like Triapine,
are reported to have cancer cell specific lethal effects (Finch et
al., 2000). It is believed that these HCTs deplete iron from key
enzymes such as ribonucleotide reductase or decrease the protein
levels of cell cycle modulators, which results in inhibition of
tumor cell growth (Le and Richardson, 2002). Based on the
structural requirement of an intact thiosemicarbazone moiety and
the iron-dependent mechanism, Compound 36 may have a similar
mechanism of action to the HCTs in inducing tumor-cell-specific
lethality. To test this hypothesis, 9 HCTs that were reported to be
active in NCI's in vivo test were collected and tested in the BJ
cell lines (FIG. 13). Some HCTs showed good selectivity between
BJ-TERT and BJ-TERT/LT/ST/RAS.sup.V12 cells. However, none was as
effective and selective as Compound 36. Moreover, the sensitivity
of the DRD cell line was never greater than in BJ-TERT cells,
implying that the activity of HCTs are generally unrelated to
oncogenic RAS signaling. This suggests some novel features of
Compound 36 unrelated to HCTs that remain to be elucidated.
##STR00081##
[0368] Compound 27 is a natural product derivative having an indole
backbone. This compound inhibited the growth of
BJ-TERT/LT/ST/RAS.sup.V12 cells at concentrations as low as 0.01
.mu.g/mL. The selectivity of Compound 27 was four-fold between
oncogenic-RAS-expressing BJ cells and non-oncogenic-RAS-expressing
BJ cells (FIG. 14A). The potency in DRD cells was the same as in
BJ-TERT/LT/ST/RAS.sup.V12 cells, indicating that Compound 27
activity depends on oncogenic RAS signaling rather than the
proliferation rate (FIG. 14A). Vi-Cell analysis confirmed Compound
27 cell growth inhibition activity and selectivity with trypan blue
exclusion (FIG. 15).
[0369] When this compound was tested in the TRE cell system,
HRAS.sup.G12V-expressing cells were slightly more sensitive than
their isogenic counterparts (FIG. 14B). This compound was active in
all oncogenic-KRAS-harboring cancer cell lines tested (FIG. 14B).
Among them, Calu-1 cells, which were derived from human lung
carcinoma, were particularly sensitive to Compound 27 (FIG. 14B).
The IC.sub.90 in Calu-1 cells was in the single-digit nanomolar
range.
[0370] Compound 27 was submitted to the Developmental Therapeutics
Program (DTP) at the National Cancer Institute (NCI) in order to
determine if there were additional cell lines, like Calu-1, that
are particularly sensitive to the compound. Compound 27 was tested
in 60 different human cancer cell lines (the NCI60 panel, see
below) and the growth inhibitory potency of Compound 27 was
determined across these cell lines. A number of cell lines were
particularly sensitive to Compound 27 treatment. These compounds
were sensitive to nanomolar concentrations and up to 10.sup.4-fold
more sensitive than the resistant cell lines (FIG. 14C and FIG.
16). Moreover, the sensitivity profiling of Compound 27 across the
60 cancer cell lines was distinct from those of known compounds in
the NCI database, as assessed by the COMPARE algorithm (Paull et
al., 1989). This suggested that the mechanism of action for
Compound 27 is unique. The kinetics of cell death induced by
Compound 27 was also uniquely rapid compared to other hit
compounds. The plasma membrane of BJ-TERT/LT/ST/RAS.sup.V12 cells
becomes permeable to trypan blue upon 8 hours of Compound 27
treatment (FIG. 14D).
[0371] To confirm that the unique properties of Compound 27 are
relevant to oncogenic-RAS-signaling pathways by knocking down KRAS
expression in oncogenic-KRAS-harboring cancer cells using RNAi,
Compound 27 was tested. Previously, Calu-1-derived cell lines were
developed: Calu-1-509 cells stably express an shRNA targeting KRAS
and Calu-1 cells are lung carcinoma cells harboring mutant KRAS
(G12C). When Compound 27 was tested in Calu-1-509 cells, they were
more resistant to Compound 27 than parental Calu-1 or Calu-1-pLKO
expressing the puromycin resistance gene without any shRNA (FIG.
14E), which confirms the involvement of oncogenic-RAS-signaling in
Compound 27-induced cell death.
[0372] Counter-screening using chemical inhibitors revealed that
GSH, L-Mim, TLCK, DFOM, SU6656, Ro31-8220, and U0126 were effective
in suppressing Compound 27-induced cell death (FIG. 17). Because
other ROS scavengers included in the list of chemical inhibitors
were not as effective as GSH, Compound 27 is likely to act as a
substrate of GSH modification that is then inactivated or excreted.
Cell death suppression by iron chelators (L-Mim and DFOM) reveals
an iron-dependent activity of Compound 27. Compound 36 also showed
iron-dependent lethality. However, the nature of iron-mediated
toxicity induced by Compound 36 and Compound 27 may be different,
because Compound 36 generates ROS and Compound 27 does not.
[0373] U0126 counter screening revealed that oncogenic RAS-RAF-MEK
signaling is important for sensitivity in Compound 27. The
non-receptor tyrosine kinase inhibitor SU6656 and the protein
kinase C inhibitor Ro31-8220 partially suppressed Compound
27-induced cell death, which reflects other aspects of RAS
signaling that contribute to sensitivity against this compound.
[0374] For Compounds 27 and 3, the IC.sub.50 value in
BJ-TERT/LT/ST/RAS.sup.V12 cells was similar to that in DRD cells,
which indicates that the synthetic lethal interaction is relevant
beyond a single cell line as shown in FIG. 42 for Compound 27, for
example. BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS.sup.V12, and DRD
cells were treated with COMPOUND 27 in 384-well plates for 48
hours. The data represents the mean.+-.SD of triplicate samples.
The results also indicate that the compounds are not
proliferation-dependent in their activity. Thus, the screening
cascade yielded two compounds, Compound 27 and Compound 3, that
have a degree of synthetic lethality with oncogenic RAS.
[0375] Regarding structure activity relationships (SAR), an analog
of Compound 27 having an unsubstituted phenyl in place of the
methyl benzoate was shown to be active. See FIG. 38. Structural
analog searching identified a compound lacking the phenyl
substituent on the backbone, but containing all other moieties
(FIG. 14A-Compound 28). This analog showed virtually the same
activity and selectivity in the BJ cell system, suggesting this
phenyl substituent is not needed. Although phenyl ring deletion may
be tolerated, some additions onto the phenyl ring and some groups
in place of the phenyl group result in loss of activity. For
example, each of the following groups in place of the methyl
benzoate of Compound 27 result in analogs having insufficient
activity:
##STR00082##
[0376] The percent growth inhibition of the imidazolyl and the
pyridinyl analogs is shown in FIG. 35, for example.
[0377] The method of preparing the biotinylated analog of Compound
27 is shown in FIG. 26 and provided in Example 5. The biotinylated
product shown, which includes the chloroacetyl group, was not
sufficiently active. This is shown in the second sheet of FIG. 26
comparing the calculated percent growth inhibition of Compound 27
to that of the bioninyated analog in a cell viability assay.
[0378] The method of making the analog having an ethynyl group at
the para position on the phenyl ring is shown in FIG. 27 and
provided in Example 5. The racemic compound had insufficient
activity. Diastereomers formed at the chiral carbon on the indole
ring attaching the para ethynyl-phenyl showed activity but lacked
selectivity, however.
[0379] Data regarding an analog having an alkyne acetate on the
phenyl ring is shown in FIG. 39, and the process for making it is
shown in Example 5.
[0380] Another analog involving the replacement of the methyl
benzoate is a compound having a --CH.sub.2C.ident.CH attached to
the indole. The method of making this compound is shown in FIG. 28.
The CH.sub.3O(C.dbd.O)CH.sub.2C.ident.CH starting material is
commercially available. DIBAL refers to diisobutyl aluminum
hydride, a reducing agent. Pictet-Spengler cyclization
proceeds.
[0381] Optional substitution on the 6-membered ring of the indole
is permissible. As noted, X is 0-4 substituent(s). When X is 1-4
substituent(s), the substituent can be independably selected from
any substituent as defined herein. FIG. 29 shows the method of
making an analog in which X is 1 and the substituent is
--OCH.sub.2C.ident.CH at the 5-indole position.
[0382] On the other hand, analogs without the chloromethyl group or
the chloro substituent alone did not inhibit cell growth at all,
emphasizing a critical role for this group (FIG. 14A--19738-13 and
FIGS. 18--19738-14). An additional 28 analogs were identified
without this chloromethyl group and they were all inactive in all
cell lines in the BJ cell system (FIG. 18).
[0383] Analogs without the methyl ester were identified. See, for
example, FIG. 14A--19738-43. The analogs showed weak activity
compared to Compound 27, indicating that the methyl ester group is
important. Analogs of Compound 27 were also tested in which the
methyl ester group was replaced with each of the following:
--C(.dbd.O)OCH.sub.2CH.sub.3; --C(.dbd.O)NH.sub.2; and
--C(.dbd.O)OCH.sub.2C.ident.CH. The percent growth inhibition for
the amide analog and the diastereomers of the ethyl analog are
shown in FIG. 34. The method of making the propargyl analog is
shown in FIG. 37, in which alkylation of Compound 5 shown therein
is the last step. Each of these analogs was found to be
insufficiently active. Extending the methyl ester by replacement
with propargyl or ethyl or converting the methyl ester to an amide
is therefore not favorable for activity.
[0384] Returning to discussion of the chloromethyl groups, the
essentiality of the chloromethyl electrophile suggests that
Compound 27 covalently labels its target protein. This should make
target identification easier, but suggests caution in translating
this compound to in vivo studies as there may be some non-specific
reactivity of this electrophilic side chain. Development candidates
would ideally have attenuated reactivity at this site. In addition,
in vivo toxicology studies may reveal whether this compound and its
analogs possess a therapeutic index. The Cl substituent is a very
good leaving group with high reactivity, although it could result
in off-target effects in vivo.
[0385] Analogs of Compound 27 were tested in which the Cl of the
chloromethyl group was replaced. The following groups in place of
Cl were found to confer insufficient activity to the analogs:
--OC(.dbd.O)CH.sub.3 which has a pKa (conjugated acid) of 4.76;
--O-tetrafluorophenyl which has a pKa (conjugated acid of <10);
and --F which has a pKa (conjugated acid) of 3.15. The percent
growth inhibition of the --O-tetrafluorophenyl,
--OC(.dbd.O)CH.sub.3, and F analogs is shown in FIG. 36. It is
noted that F has a higher electrophilicity index value than does
Cl. An acryl analog was also tested, replacing the --CH.sub.2Cl
group with an ethenyl group to form an .alpha.,.beta.-unsaturated
amide. The process of making the acryl analog is shown in FIG. 30.
The acryl analog was found to be insufficiently active, as shown in
the second sheet of FIG. 30B. In view of these results, the
nucleophile may preferably attack at the alpha carbon. The strength
of the nucleophile also appears to be limited, as the fluoro and
acetate leaving groups were not eliminated. Further analogs
include, in place of the chloromethyl group, cycloproxyl or
aldehyde, i.e., --C(.dbd.O)H, or in place of the Cl, N.sub.3.
Regarding the cyclopropoxyl analog, the epoxy amide (which
encompasses the ring N), could be attacked by the nucleophile at
the alpha or beta position. A process of making the cyclopropoxyl
analog is shown in FIG. 31. Regarding the aldehyde analog, this
group may allow for nucleophilic attack at the alpha position. A
process of making the aldehyde analog which is an oxacetamide
including the ring carbon, is shown in FIG. 32. A dicarbonyl
electrophile is used to prepare the oxoacetamide. The .alpha.-azido
compound incorporates a small group which is mild acid having a pKa
of 4.6. Replacing the Cl electrophile or the --CH.sub.2Cl group
with a group having a lower electrophilicity index value than that
of Cl (which is 3.67) is a targeted approach concerning the SAR of
the chloromethyl.
[0386] Cyclization of the (C.sub.6H.sub.4)--C(.dbd.O)OH analog of
Compound 27 may proceed by treating with mild base. The resulting
lactone is shown in FIG. 33. The electrophile is not present in the
cyclized compound.
[0387] Based on its strong potency against numerous cancer cell
lines, and its unique patterns of selectivity, Compound 27 is the
most interesting candidate out of these four scaffolds. From the
SAR studies, possible sites where affinity tags can be attached
without eliminating the activity of the compound may be inferred.
Moreover, the essentiality of the chloromethyl group suggests that
the target is covalently labeled by this compound.
Iron-Dependent Activity of Hit Compounds
[0388] Interestingly, all of these compounds, except Compound 6,
were inhibited by iron chelators, such as DFOM. Three additional
iron-binding compounds were tested for their protective effect in
order to test whether the protective effect of DFOM comes from its
ability to deplete iron or from other factors that may be specific
to DFOM. Also included in these analyses was Erastin, a previously
identified cancer-cell-specific lethal compound (Dolma et al.,
2003). The activities of Erastin, Compound 3, and Compound 36 were
significantly inhibited by all of these iron chelators (FIG. 19).
Compound 27-induced cell death was also suppressed by all of these
iron chelators moderately, indicating that its activity also
depends on iron (FIG. 19).
[0389] However, these engineered BJ tumor cells might have altered
iron content compared to their non-tumorigenic counterparts,
shifting selection pressure in the screening to identify
iron-dependent lethal compounds. To test this hypothesis, cellular
iron levels were monitored using a fluorescent iron sensor, Phen
Green SK (PGSK) (FIG. 20A; FIG. 48A). This cell-permeable molecule
has green auto-fluorescence, which diminishes upon binding to
cellular iron (Petrat et al., 2000). When the intensity of
fluorescence from PGSK-treated BJ cells was detected using flow
cytometry, BJ-TERT and BJ-TERT/LT/ST cells showed a similar
profile, whereas BJ-TERT/LT/ST/RAS.sup.V12 cells showed decreased
fluorescence. This indicated that BJ-TERT/LT/ST/RAS.sup.V12 cells
have a greater basal level of iron than its isogenic precursors
(FIG. 20A). Increased fluorescence intensity was seen in
iron-chelator-treated BJ-TERT/LT/ST/RAS.sup.V12 cells. When these
cells were treated with Compounds 3 and 27 in the presence of iron
chelators, the activities of the compounds were significantly
inhibited, highlighting the importance of iron levels for inducing
lethality (FIG. 48B).
[0390] It was hypothesized that iron-metabolism-related proteins in
BJ-TERT/LT/ST/RAS.sup.V12 cells might be adapted to allow increased
iron uptake and maintenance. Transferrin receptor 1 (TfR1) is a
membrane protein that binds to the transferrin-iron complex and is
internalized to release iron within the cytoplasm (Cheng et al.,
2004). The expression level of TfR1 was monitored by Western blot
and showed an increased amount of TfR1 expression in
BJ-TERT/LT/ST/RAS.sup.V12 cells (FIG. 20B). Thus,
BJ-TERT/LT/ST/RAS.sup.V12 cells may have greater levels of iron
because they have a greater abundance of TfR1 protein, which leads
to increased iron uptake.
[0391] A simple hypothesis as to how these hit compounds exploit
increased iron content to induce oncogenic-RAS-signal-dependent
lethality is (a) that they bind to iron to deplete the cellular
iron pool and (b) that cancer cells have higher dependency on iron,
which results in differential sensitivity to iron chelators. To
test this hypothesis, BJ-TERT/LT/ST/RAS.sup.v12 cells were treated
with these hit compounds and changes in cellular iron level were
monitored using PGSK (FIG. 20C). Treatment with control iron
chelators, DFOM and 311, induced a peak shift in the fluorescence
profile, indicating dequenching of PGSK, caused by iron depletion
(FIG. 20C (left panel)). In contrast, none of the iron-dependent
lethal compounds induced dequenching of PGSK (FIG. 20C (right
panel)).
[0392] Another indirect approach to monitor changes in iron level
upon compound treatment was used. TfR1 is important in iron
homeostasis, and it is known that the level of TfR1 protein is
tightly regulated in relation to changes in iron concentration.
Cells actively upregulate TfR1 under iron-depleted conditions
(Templeton, 2002). Treatment with control iron chelators in
BJ-TERT/LT/ST/RAS.sup.V12 cells induced an increase in TfR1 protein
level, indicating the iron regulatory machinery in
BJ-TERT/LT/ST/RAS.sup.V12 cells is normal. However, none of these
hit compounds induced TfR1 (FIGS. 20D & E). Thus, it is
unlikely that these compounds bind to and deplete cellular
iron.
[0393] BJ-TERT/LT/ST/RAS.sup.V12 cells are already engineered with
a number of antibiotic resistance genes. Accordingly, an
alternative cell line, HT1080, was used to generate TfR1 knock-down
cells using shRNAs targeted against TfR1. These TfR1 knock-down
cells were used to validate the importance of iron in
hit-compound-induced cell death by reducing iron levels using a
genetic manipulation and to compare the drug sensitivity between
the modified cell line and the parental cell line. HT1080 cells
showed good sensitivity against Compound 27 and Erastin treatment,
and shared a similar mechanistic profile: treatment with iron
chelators, MEK inhibitors or non-receptor tyrosine kinase
inhibitors effectively inhibited Compound 27-induced cell death,
while treatment with iron chelators, MEK inhibitors or antioxidants
inhibited Erastin-induced cell death, in HT1080 cells (FIG. 20F).
Erastin, Compound 3 and Compound 27 all were found to induce a
similar phenotype, RAS-RAF-MEK-dependent oxidative, iron-dependent
cell death. The bioactive compounds used suppressed cell death in
all three compounds. See data for bioactives tested for Compound 27
(FIG. 42).
[0394] shRNA expression plasmids were delivered to HT1080 cells
using a lentiviral system (Moffat et al., 2006) and puromycin was
used to generate an HT1080-derived cell line, HT1080-TfR1KD, which
stably expresses an shRNA targeting TfR1. These HT1080-TfR1KD cells
have a lower level of TfR1, determined by western blotting (FIG.
21A), and have a slightly decreased basal level of cellular iron,
determined by PGSK staining (FIG. 21B). When these iron-dependent
lethal compounds were tested in HT1080-TfR1KD cells, they showed
decreased sensitivity to Erastin, Compound 3, and Compound 36,
while parental HT1080 cells and vector-infected HT1080 cells showed
similar sensitivity to all compounds (FIG. 20G). These results
indicate that the TfR1-regulated iron pool is one of the factors
determining sensitivity of these tumor cells to
oncogenic-RAS-signal-dependent lethal compounds.
[0395] To validate the importance of iron in lethal
compound-induced cell death, iron levels were reduced using a
genetic method, and then drug sensitivity was compared between the
modified cell line and the parental cell line. Three different
shRNAs targeting TfR1 were delivered to BJ-TERT/LT/ST/RAS.sup.V12
cells using a lentiviral system (Moffat et al., 2006). Two shRNA
clones displayed moderate level of knock down efficiency (clone 658
and 659) whereas one shRNA clone was not effective, as assessed by
western blot analysis (clone 660) (FIG. 48C). When erastin was
tested in these cells, erastin became less effective in
clone-658-expressing and clone-659-expressing cells, compared to
clone-660-expressing and non-targeting-shRNA-expressing cells.
However, the level of cell death suppression was not comparable to
that of iron-chelator-treated cells. This partial rescuing activity
of shRNAs targeting TfR1 may reflect insufficient level of knock
down or the existence of other mechanisms for enriching the iron
content of RAS-transformed cells.
[0396] To explore other aspects of oncogenic-RAS-signaling relevant
to iron enrichment, the expression level of other genes whose
functions are known to modulate cellular iron content was compared.
The TfR1-mediated pathway is one route of iron uptake, but the
cellular iron pool is also controlled by an iron storage protein
complex, consisting of ferritin heavy chain 1 (FTH1) and ferritin
light chain (FTL). (Harrison and Arosio, 1996). Real-time RT-PCR
analysis showed a gradual increase in TfR1 mRNA levels across the
BJ-TERT, BJ-TERT/LT/ST and BJ-TERT/LT/ST/RAS.sup.V12 cells, in
agreement with western blot analysis (FIG. 48D and FIG. 20B).
Interestingly, the expression level of both FTH1 and FTL was
specifically decreased in BJ-TERT/LT/ST/RAS.sup.V12 cells (FIGS.
48E and 48F), indicating that oncogenic-RAS-signaling has a dual
mechanism to augment the cellular labile iron pool: one is to
increase iron uptake via up-regulation of TfR1, the other is to
reduce the capacity of iron storage via down-regulation of FTH1 and
FTL.
Involvement of VDAC3 in Erastin-Induced and Compound 3-Induced Cell
Death
[0397] As erastin, Compound 3, and Compound 27 emerged from a
similar screening setting, and share a common genetic selectivity
and induced phenotype, whether Compound 3 and Compound 5 act
through the same mechanism as erastin, by engaging mitochondrial
VDACs was replaced. Using affinity purification and mass
spectrometry, mitochondrial voltage-dependent anion channels
(VDACs) were identified as specific targets of erastin (Yagoda et
al., 2007).
[0398] Mammalian cells have three VDAC isoforms, VDAC1, VDAC2 and
VDAC3. An erastin affinity analog specifically purifies VDAC2 and
VDAC3 only in lysates of BJ-TERT/LT/ST/RAS.sup.V12 cells, not of
BJ-TERT cells. RNA interference experiments targeting VDAC2 and
VDAC3 demonstrated that erastin binding to VDAC2 and VDAC3 induces
a gain-of-function lethality: knocking down VDAC3 makes cells
resistant to erastin, and knocking down VDAC2 reduces their
sensitivity to erastin (Yagoda et al., 2007).
[0399] To test the possibility of VDAC involvement in the synthetic
lethal action of Compound 3 and Compound 27, VDAC3 in HT1080 cells
were knocked down, using short hairpin RNAs (shRNAs) specifically
targeting VDAC3. Knockdown of VDAC3 protein suppressed
erastin-induced and Compound 3-induced cell death, whereas Compound
27 lethality was not affected (FIG. 43). Introduction of shRNAs
targeting VDAC3 did not change the level of other VDAC isoforms
(VDAC1 and VDAC2), as assessed by quantitative PCR (Q-PCR) (FIG.
44). Thus, Compound 3 acts through at least VDAC3, as does erastin.
However, Compound 27 appears to act in a VDAC3-independent
manner.
[0400] This is of interest, because it demonstrates that the
oxidative, non-apoptotic cell death pathway targeted by these
compounds is not restricted to VDAC involvement, but can be
activated under other conditions and may be more generally
important.
Compound 27 has Oncogenic-RAS-Signal Dependent Lethality with a
Unique Sensitivity Profile
[0401] Compound 27's ability to induce synthetic lethality with
oncogenic RAS was rapid and quite potent. This compound inhibited
the growth of BJ-TERT/LT/ST/RAS.sup.V12 and DRD cells as low as 10
ng/mL (FIG. 42 panel A) and started to kill sensitive cells as
early as 8 hours after treatment (FIG. 45 panels A and C). A trypan
blue exclusion assay was carried out to confirm the growth
inhibitory effect and the selectivity of Compound 27. This was
performed in a Vi-Cell (Beckman Coulter), which stains cells with
trypan blue, takes 100 images, and analyzes the images to calculate
the number of viable cells (i.e. cells excluding trypan blue). This
assay eliminates false positives caused by auto-fluorescence or
redox activity from a compound in the alamar blue assay. The trypan
blue exclusion assays with Compound 27 revealed a similar level of
potency and selectivity as the alamar blue assay (FIG. 45 panel B).
Moreover, longer treatment had little effect on the viability of
cells lacking oncogenic RAS, confirming the qualitative nature of
Compound 27's selectivity (FIG. 45 panel B).
[0402] Cell death induced by Compound 27 treatment was not blocked
by pan-caspase inhibitors (z-VAD-fmk, Boc-D-fmk), suggesting that a
caspase-independent death pathway is involved in Compound
27-induced cell death. Because the survival effect of caspase
inhibitors can be masked by activation of alternative cell death
pathways upon drug treatment, we employed a more sensitive
measurement of caspase activation to examine the possible
involvement of caspases in Compound 27-induced cell death.
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear
enzyme that is subject to cleavage by activated caspase 3/7.
Staurosporine, erastin, Compound 3 and Compound 5 treatments of
BJ-TERT/LT/ST/RAS.sup.V12 cells were compared, cell lysates were
prepared, PARP-1 cleavage was analyzed by western blot with an
anti-PARP-1 antibody (FIG. 45 panel B). Only staurosporine-treated
sample showed PARP-1 cleavage, indicating that staurosporine
induces classical apoptotic cell death involving caspases, whereas
erastin, Compound 3, and Compound 27 activate a non-apoptotic
pathway (FIG. 45 panel B).
[0403] Among the three RAS genes (HRAS, KRAS, NRAS), the BJ cell
system employed HRAS mutations to initiate oncogenic signals. All
three RAS protein can activate common downstream effectors but, for
elusive reasons, human cancer cells have biased mutation rates
among the three RAS genes; more than 70% contain KRAS mutations,
25% NRAS mutations and less than 5% HRAS mutations (Rodenhuis,
1992). Since KRAS mutations are the most prevalent in human
cancers, the activity of Compound 27 in KRAS-mutation-harboring
cancer cells (FIG. 46 panel A). This compound was active in all
oncogenic-KRAS-harboring cancer cell lines tested (FIG. 46 panel
A). Among them, Calu-1 cells were the most sensitive to Compound 27
(FIG. 46 panel A); the IC.sub.50 in Calu-1 cells was 20 ng/mL.
[0404] Considering Compound 27's high potency, the compound was
tested in a broader range of cancer cell lines to see if additional
cell lines, like Calu-1, are particularly sensitive. Compound 27
was tested in 60 different human cancer cell lines (the NCI60
panel) and the growth inhibitory potency of Compound 27 was
determined across these cell lines. A number of cell lines were
particularly sensitive to Compound 27 treatment; nanomolar
concentrations of Compound 27 were often sufficient to induce
growth arrest or cell killing, and there was up to 10.sup.4-fold
differences in sensitivity between resistant and sensitive cell
lines (FIG. 46 panel B). Moreover, the sensitivity profile of
Compound 27 across the 60 cancer cell lines was distinct from those
of known compounds in the NCI database, as assessed by the COMPARE
algorithm (Paull et al., 1989), which suggests the mechanism of
action for Compound 27 is unique (FIG. 46B). The COMPARE algorithm
compares sensitivity profiles of an input compound to those of all
other compounds in the database, and returns a similarity score in
terms of Pairwise Correlation Coefficient (PCC). In control tests,
the sensitivity profile of paclitaxel grouped with vinblastine,
maytansine and rhizoxin, with high PCC values (FIG. 46B). All of
these compounds are known to perturb microtubule integrity, which
demonstrates the usefulness of the COMPARE algorithm to understand
the mechanism of compound action. In contrast, the COMPARE analysis
of Compound 3 returned only compounds with low PCC values (FIG.
46B). In other words, no compounds in the NCI database had a
similar pattern of cell killing as Compound 27.
[0405] It was inferred that the oncogenic RAS-signal-dependent
lethality of Compound 27 should persist in cancer cell lines other
than HRAS-transformed fibroblasts, because the protein target of
Compound 27 is not HRAS itself, but is a protein associated with
the oncogenic RAS network whose status changes upon activation of
oncogenic RAS signaling, regardless of subtype of RAS (H--, N--, or
K-RAS) or tissue origin (sarcomas or carcinomas). To test this
hypothesis, Calu-1 cells were cultured which were derived from a
human lung carcinoma, and attenuated oncogenic RAS signaling using
shRNAs targeting HRAS, NRAS or KRAS. Compound 27 was less effective
in Calu-1 cells expressing shRNAs targeting each RAS isoform,
regardless of subtype (FIG. 46C). Treatment with shRNAs targeting
RAS isoforms did not lead to a general survival effect, since loss
of sensitivity to staurosporine was not observed. (FIG. 47).
Example 2
[0406] Lack of potency and non-specific toxicity are the major
hurdles in anti-cancer drug development. Traditionally, these
features were not examined until the very late phase of drug
development. Ideally, efforts to address the two properties should
be made in early stage of drug discovery and only drug candidates
that show consistent and robust activity should be put forward to
improve overall efficiency.
[0407] NCI60 anti-cancer screening ("NCI60 test") is a system that
provides an opportunity to assess a drug candidate's potency and
selectivity at a relatively early stage of drug discovery. The
primary purpose of NCI60 anti-cancer screening is to identify
lethal compounds that have significantly increased potency against
some selective cancer cell lines. Such compounds would be expected
to kill sensitive cancer cells while having minimal effect on
healthy cells in a predetermined concentration range. NCI60
anti-cancer screening makes use of 60 cancer cell lines from
different human tissue origins. Cells are seeded on a 96-well
plate, treated with lethal compounds in 5-point, 10-fold dilution
series pattern starting at 100 .mu.M (i.e. 10 nM, 100 nM, 1 .mu.M,
10 .mu.M, 100 .mu.M) for 2 days, and measured for cell viability
using sulforhodamine B dye. The optical densities of treated
samples are determined using a plate reader and percent growth
value is calculated by processing the optical density value.
[0408] Three parameters are used in determining potency and
selectivity of a given test agent from the NCI60 anti-cancer
screening data: GI.sub.50, TGI, and LC.sub.50. GI.sub.50 is the
concentration of test agent which inhibits cell growth by 50%,
calculated as follows:
100.times.(T-T.sub.0)/(C-T.sub.0)=50
wherein T is the optical density of test well after 2-days of
treatment, T.sub.0 is the optical density at time 0, and C is the
optical density of a control well without a test agent.
[0409] Thus, GI.sub.50 measures the growth inhibitory power of a
test agent.
[0410] TGI is the concentration of a test agent which completely
inhibits cell growth, calculated as follows:
100.times.(T-T.sub.0)/(C-T.sub.0)=0
wherein T is the optical density of test well after 2-days of
treatment, T.sub.0 is the optical density at time 0, and C is the
optical density of a control well without a test agent.
[0411] Thus, TGI measures the cytostatic effect of a test
agent.
[0412] The LC.sub.50 is the concentration of a test agent that
causes the death of 50% of the cells, calculated as follows:
100.times.(T-T.sub.0)/T.sub.0=-50
wherein T is the optical density of test well after 2-days of
treatment, and T.sub.0 is the optical density at time 0.
[0413] Thus, LC.sub.50 measures the lethal effect of a test
agent.
[0414] The entire NCI60 test is replicated at least twice to ensure
consistency of the data and to determine accurate GI.sub.50, TGI
and LC.sub.50 values. The panel of results for each candidate
compound in the NCI60 test is known as the "sensitivity
profile."
[0415] Based on our past experience, test agents (i.e., candidate
compounds) with nanomolar (nM) range TGI or LC.sub.50 values are
considered to have enough potency for in vivo testing. If the
sensitivity of some cancer cell lines is greater than average of
all 60 cell lines, then, the test agent can be expected to be able
to induce selective cell killing. Moreover, if the sensitivity
profile of a test agent across the 60 cell lines is distinct from
that of known anti-cancer reagents, the test agent can be
considered a novel class of anti-cancer agent.
[0416] In comparing sensitivity profiling, the COMPARE analysis was
conducted, which was developed from NCI. The NCI provides a COMPARE
server for users to input the GI.sub.50, TGI, or LC.sub.50 values
of their test compound as a `seed` for COMPARE analysis. The server
then compares the sensitivity pattern of the `seed` to those in
their database consisting of sensitivity profiling data from
>100,000 compounds and sends out the result of COMPARE analysis
by displaying rank-ordered lists of compounds. To derive COMPARE
rankings, an index of similarity called "pair-wise correlation
coefficient" (PCC) was used. In general, a PCC value greater than
0.7 indicates the same mechanism of action or the same target of
the two compounds.
[0417] Erastin (Table 2; FIG. 22), and Compounds 3 (Table 3; FIG.
25), 6 (Table 4; FIG. 24), 27 (Table 6; FIG. 16), and 36 (Table 5;
FIG. 23) were submitted for NCI60 testing. The results of the NCI60
screening of Erastin and Compounds 3, 6, 27, and 36 are summarized
in Tables 2-6. A GI.sub.50 concentration below 10 .mu.M was
reported as sensitive. The tumor type, cell line, GI.sub.50, TGI,
and LC.sub.50, and RAS mutation (if any) are also summarized.
TABLE-US-00002 TABLE 2 Cancer cell lines sensitive to Erastin
identified by NCI60. ERASTIN - NCI60 GI 50% GI 100% LC 50% Tumor
Type Cell Line (.mu.M) (.mu.M) (.mu.M) RAS Mutation Renal 786-0
2.03 4.12 8.36 Cancer ACHN 0.979 2.5 6.3 CAKI-1 2.15 n.d. >10
RXF 393 2.16 7.2 >10 UO-31 0.373 1.63 >10 Leukemia HL-60 2.26
>10 >10 NRAS Q61L K-562 3.39 >10 >10
TABLE-US-00003 TABLE 3 Cancer cell lines sensitive to Compound 3
identified by NCI60. Compound 3 - NCI60 GI GI 50% 100% LC 50% RAS
Tumor Type Cell Line (.mu.M) (.mu.M) (.mu.M) Mutation Leukemia
CCRF-CEM 7.95 >10 >10 KRAS G12D HL-60(TB) 9.8 >10 >10
NRAS Q61L K-562 9.16 >10 >10 MOLT-4 8.39 >10 >10 NRAS
Q61L SR 7.15 >10 >10 Non-small lung HOP-92 6.18 >10 >10
carcinoma Colon Cancer HT29 9.82 >10 >10 CNS Cancer SNB-75
9.61 >10 >10 Renal Cancer 786-0 9.27 >10 >10 A498 3.39
>10 >10 UO-31 6.82 >10 >10 Breast Cancer NCI/ADR-RES
8.93 >10 >10 Hs578T 9.31 >10 >10 HRAS G12D BT-549 4.28
>10 >10
TABLE-US-00004 TABLE 4 Cancer cell lines sensitive to Compound 6
identified by NCI60. Compound 6 - NCI60 GI 50% GI 100% LC 50% Tumor
Type Cell Line (.mu.M) (.mu.M) (.mu.M) RAS Mutation Leukemia
CCRF-CEM 0.115 0.422 n.d. KRAS G12D HL-60(TB) 0.516 2.38 8.25 NRAS
Q61L K-562 0.238 >10 MOLT-4 0.783 3.91 >10 NRAS Q61L
RPMI-8226 2.37 6.77 >10 KRAS G12A SR 2.65 8.4 >10 Non-small
cell HOP-92 1.48 4.31 >10 lung carcinoma NCI-H23 4.17 >10
>10 KRAS G12C NCI-H522 0.49 1.79 5.12 Colon Cancer COLO 205
0.232 1.26 4.78 HCT-116 0.376 1.32 3.64 KRAS G13D HT29 3.32 >10
>10 KM12 5.2 >10 >10 SW-620 0.271 1.03 3.9 KRAS G12V CNS
Cancer SF-268 2.46 7.71 >10 SF-539 2.32 4.71 9.59 Melanoma LOX
IMVI 1.73 3.29 6.25 MALME-3M 0.261 0.723 2.8 M14 2.07 4.38 9.28
SK-MEL-2 2.32 6.87 >10 NRAS Q16R SK-MEL-28 1.46 3.11 6.63
SK-MEL-5 5 >10 >10 UACC-257 2.78 >10 >10 UACC-62 9.78
>10 >10 Ovarian OVCAR-3 3.52 >10 >10 Cancer OVCAR-4
5.71 >10 >10 OVCAR-8 4.22 >10 >10 Renal Cancer 786-0
2.51 5.54 >10 ACHN 8.76 >10 >10 RXF393 0.652 2.41 6.6
SN12C 8.1 >10 >10 TK-10 2.76 >10 >10 Prostate PC-3 5.07
>10 >10 Cancer DU-145 5.28 >10 >10 Breast Cancer MCF-7
1.55 3.78 9.18 MDA-MB-231/ATCC 1.75 4.09 9.52 KRAS G13D HS 578T
3.31 >10 >10 HRAS G12D MDA-MB-435 2.21 4.38 9.07 BT-549 1.49
3.27 7.21 T-47D 4.53 2.06 9.35
TABLE-US-00005 TABLE 5 Cancer cell lines sensitive to Compound 36
identified by NCI60. Compound 36 - NCI60 GI 50% GI 100% LC 50%
Tumor Type Cell Line (.mu.M) (.mu.M) (.mu.M) RAS Mutation Leukemia
CCRF-CEM 0.165 0.705 5.57 KRAS G12D HL-60(TB) 0.163 0.545 2.62 NRAS
Q61L K-562 0.267 1.08 3.51 MOLT-4 0.0773 1.1 7.39 NRAS Q61L
RPMI-8226 0.194 0.889 6.98 KRAS G12A SR 1.32 3.4 8.78 Non-small
cell A549/ATCC 1.11 2.4 5.15 KRAS G12S lung carcinoma EKVX 1.24
2.51 5.09 HOP-62 0.405 1.42 3.77 KRAS G12C HOP-92 0.479 1.52 3.96
NCI-H226 0.48 1.66 4.55 NCI-H23 0.44 1.65 4.23 KRAS G12C NCI-H322M
0.606 1.99 4.93 NCI-H460 0.626 2.02 5.02 KRAS Q61H NCI-H522 0.0829
0.309 1.18 Colon Cancer COLO 205 0.184 0.451 1.46 HCC-2998 0.24
0.733 2.68 KRAS A146T HCT-116 0.252 1.11 3.32 KRAS G13D HCT-15
0.305 1.53 3.93 KRAS G13D HT29 0.322 1.37 4.1 KM12 0.469 1.8 4.47
SW-620 0.184 0.569 2.54 KRAS G12V CNS Cancer SF-268 1.09 2.5 5.74
SF-295 1.19 2.52 5.33 SF-539 0.191 0.349 0.638 SNB-19 1.05 2.29 5
SNB-75 0.372 1.26 4.3 U251 0.47 1.7 4.12 Melanoma LOX IMVI 0.203
0.478 1.32 MALME-3M 0.55 1.83 4.57 M14 0.384 1.59 4.8 SK-MEL-2
0.297 1.01 4.93 NRAS Q16R SK-MEL-28 0.239 0.592 2.07 SK-MEL-5 0.209
0.898 3.05 UACC-257 1.26 2.97 6.99 UACC-62 0.214 0.427 0.853
Ovarian IGROV1 0.254 1.28 4.17 Cancer OVCAR-3 0.204 0.804 4.2
OVCAR-4 0.147 0.405 1.31 OVCAR-5 0.492 1.62 4.33 KRAS G12V OVCAR-8
1.16 2.94 7.48 SK-OV-3 1.26 2.56 5.2 Renal Cancer 786-0 0.252 0.907
3.02 A498 0.268 1.29 3.85 ACHN 0.366 1.43 3.78 CAKI-1 0.165 0.413
1.08 RXF393 0.181 0.485 2.49 SN12C 0.351 1.35 3.67 TK-10 0.296 1.08
3.47 UO-31 0.125 0.385 1.47 Prostate PC-3 0.548 1.82 4.26 Cancer
DU-145 1.14 2.35 4.84 Breast Cancer MCF-7 0.265 1.34 3.83
NCI/ADR-RES 1.04 2.34 5.27 MDA-MB-231/ATCC 0.341 1.36 3.27 KRAS
G13D HS 578T 0.42 2.27 >10 HRAS G12D MDA-MB-435 0.175 0.781 3
BT-549 0.302 1.3 4.07 T-47D 0.701 3.21 >10
TABLE-US-00006 TABLE 6 Cancer cell lines sensitive to Compound 27
identified by NCI60. Compound 27 - NCI60 GI 50% GI 100% LC 50%
Tumor Type Cell Line (.mu.M) (.mu.M) (.mu.M) RAS Mutation Leukemia
CCRF-CEM 0.0195 0.0431 0.0953 KRAS G12D HL-60(TB) 0.28 0.537 1.2
NRAS Q61L MOLT-4 1.02 2.74 7.39 NRAS Q61L RPMI-8226 0.0431 2.22
6.99 KRAS G12A SR 0.4 1.6 6.58 Non-small cell A549/ATCC 2.51 >10
>10 KRAS G12S lung carcinoma EKVX 2.31 5.63 >10 HOP-92 0.0991
1.07 5.85 NCI-H226 0.688 2.79 >10 NCI-H23 1.15 2.95 7.59 KRAS
G12C NCI-H322M 3.6 >10 >10 NCI-H460 1.43 3.11 6.74 KRAS Q61H
NCI-H522 1.63 3.74 8.58 Colon Cancer COLO 205 1.67 3.44 7.07
HCC-2998 2.01 5.1 >10 KRAS A146T HCT-116 0.364 1.48 3.84 KRAS
G13D HCT-15 1.45 2.83 5.54 KRAS G13D HT29 0.415 1.46 4.06 KM12
0.188 1.19 4.15 SW-620 0.681 2.09 5.25 KRAS G12V CNS Cancer SF-268
0.0692 0.218 0.577 SF-295 1.1 2.97 8.06 SF-539 0.118 0.241 0.492
SNB-19 2.35 7.41 >10 SNB-75 0.745 3.06 >10 U251 1.1 2.3 4.79
Melanoma LOX IMVI 0.182 0.387 0.826 MALME-3M 0.406 1.53 4.6 M14
1.07 2.25 4.75 SK-MEL-2 1.74 4.13 9.82 NRAS Q16R SK-MEL-28 1.62
3.31 6.76 SK-MEL-5 1.38 2.67 5.16 UACC-257 1.37 7.46 >10 UACC-62
1.21 2.47 5.04 Ovarian IGROV1 0.0209 0.0571 0.27 Cancer OVCAR-3
0.353 1.57 6.59 OVCAR-4 4.3 >10 >10 OVCAR-5 2.24 5.71 >10
KRAS G12V OVCAR-8 2.32 >10 >10 SK-OV-3 1.68 3.34 6.67 Renal
Cancer 786-0 0.0603 0.473 2.52 A498 1.83 4.52 >10 ACHN 0.187
1.05 3.25 CAKI-1 0.0211 0.0381 0.0687 RXF393 1.3 3 6.93 SN12C 1.25
2.77 6.12 TK-10 1.9 3.96 8.25 UO-31 0.02 0.0385 0.0743 Prostate
PC-3 0.231 1.08 3.29 Cancer DU-145 0.308 1.4 4.01 Breast Cancer
MCF-7 0.782 3.98 >10 NCI/ADR-RES 2.56 >10 >10
MDA-MB-231/ATCC 1.2 3.32 9.18 KRAS G13D HS 578T 0.403 2.85 >10
HRAS G12D MDA-MB-435 1.71 3.74 8.19 BT-549 0.131 0.316 0.765 T-47D
1.64 5.16 >10
[0418] When considering the potency of the NCI60-tested compounds,
Compound 27 was the only one that showed a nanomolar (nM)
concentration range of cell killing activity. Both Erastin (Table
2; FIG. 22) and Compound 27 (Table 6; FIG. 16) showed interesting
selectivity against some specific cancer cell lines. Indeed,
Erastin showed selectivity against tissue origin--renal cancer
cells are more sensitive to Erastin treatment (FIG. 22). The
COMPARE analysis was conducted to determine whether Erastin or
Compound 27 have overlapping sensitivity profiling with any of the
compounds in the database. However, the highest PCC scores of
Erastin or Compound 27 were 0.4 and 0.5 respectively using TGI
value as an input, which implies that the mechanism of action or
target protein of these two compounds are novel ones.
[0419] The sensitivity profile data from NCI60 anti-cancer
screening can also be used for biomarker identification when it is
combined with expression profile data of the 60 cancer cell lines.
cDNA microarray data of NCI60 cancer cell lines are available from
public resources. The expression profile of 9,706 genes in the 60
NCI cell lines was downloaded and a search was carried out for
genes that are over-expressed only in sensitive cell lines upon
Erastin or Compound 27 treatment. The cut-off value was set at
10-fold greater than the average amount of mRNA. Several genes were
identified that are specifically up-regulated in Erastin sensitive
cell lines (Table 6B). The top hit gene was PDGF-B polypeptide,
which recaptured the oncogenic-RAS-signal dependent lethality of
Erastin because RAS is one of the major downstream mediators of
PDGF/PDGFR signaling. Human protein tyrosine phosphatase 1E (PTP1E)
and phospholipase C beta 4 are among the top hit genes and they are
involved in the PDGF/PDGFR signaling directly or indirectly
suggesting that status of PDGF/PDGFR signaling can be an important
biomarker for Erastin sensitivity.
TABLE-US-00007 TABLE 6B Results of biomarker identification using
sensitivity profile data from NCI60 anti-cancer screening in
combination with expression profile data of the 60 cancer cell
lines. % % Sensitive Resistant Gene Name 100 5 Platelet-derived
growth factor beta polypeptide (simian sarcoma viral (v-sis)
oncogene homolog) 100 7 Aldehyde reductase 1 (low Km aldose
reductase) 100 7 ESTs, Weakly similar to KIAA0108 100 7 Human
protein tyrosine phosphatase 1E (PTP1E) 100 9 Crystallin zeta
(quinone reductase) 100 11 Phospholipase C, beta 4 100 11 Unknown
100 11 Unknown 100 11 Aldehyde reductase 1 (low Km aldose
reductase) 67 22 SID 272525, ESTs [5':, 3': N35886] 56 16 AXL AXL
receptor tyrosine ki0se Chr.19 [112500, (DW), 5': T85905, 3':
T91043] 56 18 SID 42787, ESTs 5': R59827, 3': R59717] 56 24 ESTs
Chr.6 [154211, (DI), 5': R51950, 3': R53619] 56 39 SID 49722, ESTs
[5': H29102, 3': H28999] 56 24 SID 236277, ESTs [5': H61273, 3':
H61274] 56 20 SID 486215, Uroki0se-type plasminogen activator [5':,
3': AA040727] 56 33 SID W 486055, Ctyochome P450 IB1
(dioxin-inducible) [5': AA043141, 3': AA040872] 56 20 Human LOT1
mR0, complete cds Chr.6 [486715, (I), 5':, 3': AA043116] 56 51 SID
W 510056, Homo sapiens (clone zap128) mR0, 3' end of cds [5':
AA053065, 3': AA053409]
[0420] The results of the NCI60 test demonstrates that Compound 27
has a unique sensitivity profile across the 60 human cancer cell
line panel and a very potent lethality for some selective cancer
cell lines. The sensitive cell lines are: SF-539, LOX, IGROV1,
CAKI-1, UO-31, BT-549, CCRF-CEM, 786-0, HOP-92, PC-3, DU-145,
NCI-H522, SF-268, SF-295, and SNB-75. Any lethal compound that
shows a similar sensitivity profile in the NCI60 test can be
considered to have a similar mechanism of action. A plot of the
percent growth vs. log concentration for Compound 27 in the NCI60
test in all cell lines of Table 6 is shown in FIG. 40.
[0421] In addition, Erastin and Compounds 3, 6, 27, and 36 were
tested to determine their potency and selectivity for additional
cell lines using the Alamar Blue assay (see above). The results of
this additional testing are summarized in Tables 7-10 (no
additional cell lines were identified as sensitive for Compound 3).
If a concentration of below 10 .mu.g/ml of the compound inhibited
the cancer cell line's growth by 50 percent, the cell line was
reported as sensitive. The tumor type, cell line, maximum
percentage inhibition, the concentration of compound required to
inhibit cell growth by 50%, and RAS mutation (if any) are also
summarized.
TABLE-US-00008 TABLE 7 Additional cancer cell lines sensitive to
Erastin identified by Alamar Blue assay. ERASTIN - ALAMAR BLUE
ASSAY Maximum GI 50% RAS Tumor Type Cell Line Inhibition % (.mu.M)
Mutation Foreskin fibroblasts w/ TERT, BJELR 100 6 HRAS LT, ST, RAS
G12V Leimyosarcoma, vulva SK-LMS-1 100 6 Uterine sarcoma MES-CA 100
3 Fibrosarcoma HT1080 98 2 NRAS Q16K Ewing sarcoma, anaplastic
SK-ES-1 98 7 osteosarcoma Osteosarcoma U-2 OS 96 6 Cervical
carcinoma HeLa 94 0.6 Ewing sarcoma TC71 92 10 Peripheral
neuroepithelioma TC32 88 8 Acute myelogenous leukemia U973 73 10
Uterine, mixed mesodermal SK-UT 73 4 tumor Non-small cell lung
carcinoma Calu-1 100 6 KRAS G12C
TABLE-US-00009 TABLE 8 Additional cancer cell lines sensitive to
Compound 6 identified by Alamar Blue assay. Compound 6 - ALAMAR
BLUE ASSAY Maximum GI 50% RAS Tumor Type Cell Line Inhibition %
(.mu.M) Mutation Pancreatic Cancer PaCa-2 100 0.6 HRAS G12C
Non-small cell lung Calu-1 100 2.5 KRAS carcinoma G12C
TABLE-US-00010 TABLE 9 Additional cancer cell lines sensitive to
Compound 36 identified by Alamar Blue assay. Compound 36 - ALAMAR
BLUE ASSAY Maximum GI 50% RAS Tumor Type Cell Line Inhibition %
(.mu.M) Mutation Pancreatic Cancer PaCa-2 100 1.5 HRAS G12C
Non-small cell lung Calu-1 100 0.3 KRAS carcinoma G12C
TABLE-US-00011 TABLE 10 Additional cancer cell lines sensitive to
Compound 27 identified by Alamar Blue assay. Compound 27 - ALAMAR
BLUE ASSAY Maximum GI 50% RAS Tumor Type Cell Line Inhibition %
(.mu.M) Mutation Pancreatic Cancer PaCa-2 100 0.125 HRAS G12C
Non-small cell lung Calu-1 100 0.001 KRAS carcinoma G12C
TABLE-US-00012 TABLE 11 Sequence of Primers for Q-PCR. Sequence of
primers used in real-time quantitative PCR Description Sequence
Identification VDAC1 F: 5'-CCTGGACAGCAGGAAACAGTA SEQ ID NO.: 1
AC-3' VDAC1 R: 5'-AGGCGTCAGGGTCAATCTG SEQ ID NO.: 2 A-3' VDAC2 F:
5'-TGATTTTGCTGGACCTGCA SEQ ID NO.: 3 A-3' VDAC2 R:
5'-CAGCAAGCCAGCCCTCAT-3' SEQ ID NO.: 4 VDAC3 F:
5'-AATTTCGCCCTGGGTTACA SEQ ID NO.: 5 A-3' VDAC3 R:
5'-TCAGTGCCATCGTTCACATG SEQ ID NO.: 6 T-3' TfR1 F:
5'-GAAAACAGACAGATTTGTCAT SEQ ID NO.: 7 G-3' TfR1 R:
5'-CTCTTTTGGAGATACGTAGG SEQ ID NO.: 8 G-3' DMT1 F:
5'-CATCACTATTATGGCCCTCA SEQ ID NO.: 9 C-3' DMT1 R:
5'-GAACATGCCCTTGAGTACCT SEQ ID NO.: 10 G-3' HCP1 F:
5'-GGTCTTTGCCTTTGCCACTAT SEQ ID NO.: 11 C-3' HCP1 R:
5'-CAGGTGTGATGACTAATGACA SEQ ID NO.: 12 GG-3' FTH1 F:
5'-CAGATCAACCTGGAGCTCTA SEQ ID NO.: 13 C-3' FTH1 R:
5'-CTTCAAAGCCACATCATCG SEQ ID NO.: 14 C-3' FTL F:
5'-GGCCCTGGAGAAAAAGC-3' SEQ ID NO.: 15 FTL R: 5'-GAAGTGAGTCTCCAGGAA
SEQ ID NO.: 16 G-3' RPLP0 F: 5'-ACGGGTACAAACGAGTCCT SEQ ID NO.: 17
G-3' RPLP0 R: 5'-GCCTTGACCTTTTCAGCAA SEQ ID NO.: 18 G-3'
Example 3
[0422] Below is a representative synthesis for compounds of formula
IV. In particular, the synthesis of compound 27 is set forth
below:
Synthesis of methyl esters of L- and D-tryptophanes
chlorohydrates
##STR00083##
[0424] Thionylchloride (0.064 mol) was slowly added to a cooled
(0.degree. C.) suspension of tryptophane (0.049 mol) in methanol
(150 mL). The reaction mixture was warmed up to 40.degree. C. and
stirred at this temperature for six hours. All solvents were
removed and the solid residue was triturated with ether. The solid
was filtered off to give the required product.
[0425] D-tryptophane (2a): yield 98%, M.p. 232-233.degree. C.
NMR.sup.1H (.delta., ppm, DMSO-d6, 300 MHz): 3.39 (2H, m,
CH.sub.2); 3.63 (3H, s, CH.sub.3O); 4.20 (1H, t, CH, J.sub.HH=5.5
Hz); 7.07 (2H, dt, Ar, J.sub.HH=21 Hz, 6 Hz); 7.26 (1H, d, H.sub.2,
J.sub.HH=3 Hz); 7.39 (1H, d, Ar, J.sub.HH=7.8 Hz); 7.53 (1H, d, Ar,
J.sub.HH=7.8 Hz). NMR.sup.13C (.delta., ppm, DMSO-d6, 125.76 MHz):
26.01 (s), 52.61 (d), 106.26 (s), 111.45 (s), 117.84 (s), 118.50
(s), 121.05 (s), 124.80 (s), 126.79 (s), 136.13 (s), 169.57 (s).
m/z 218 (M.sup.+).
[0426] L-tryptophane (2b): yield 98%, M.p. 225-226.degree. C.
NMR.sup.1H (.delta., ppm, DMSO-d6, 300 MHz): 3.29 (2H, m,
CH.sub.2); 3.62 (3H, s, CH.sub.3O); 4.18 (1H, t, CH, J.sub.HH=5.5
Hz); 6.99 (1H, t, Ar, J.sub.HH=4.5 Hz); 7.07 (1H, t, Ar,
J.sub.HH=4.5 Hz); 7.25 (1H, d, H.sub.2, J.sub.HH=3 Hz); 7.37 (1H,
d, Ar, J.sub.HH=4.8 Hz); 7.51 (1H, d, Ar, J.sub.HH=4.8 Hz).
NMR.sup.13C (.delta., ppm, DMSO-d6, 125.76 MHz): 25.94 (s), 52.85
(d), 106.33 (s), 111.08 (s), 117.86 (s), 118.44 (s), 120.97 (s),
124.78 (s), 126.81 (s), 136.12 (s), 169.42 (s). m/z 218
(M.sup.+).
Free-Base of Compounds 2a and 2b:
##STR00084##
[0428] Triethylamine (0.055 mol) was added to a suspension of the
corresponding hydrochloride 2a or 2b (0.05 mol) in benzene (100 mL)
at room temperature. The mixture was stirred for one hour and then
filtered. The filtrate was concentrated to give the product as a
clear oil, which was dried under a high vacuum for ten minutes. The
product was immediately used for the following reaction:
Synthesis of (3S)- and
(3R)-methyl-1-(p-carbmethoxyphenyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-i-
ndole-3-carboxylates
##STR00085##
[0430] 4 .ANG. molecular sieves (2 g) and trifluoroacetic acid (0.5
mL) were added to a solution of 3a or 3b (0.06 mol) and methyl
benzaldehyde-4-carboxylate (0.05 mol) in benzene (150 mL). The
reaction mixture was stirred at reflux for one hour and additional
trifluoroacetic acid (0.138 mol) was added. The reaction mixture
was refluxed for three more hours and then cooled to room
temperature. Water (50 mL) was added and the resulting mixture was
basified to a pH of 8 with 30% NaOH in water. The layers were
separated. The organic layer was dried with MgSO.sub.4,
concentrated to about one quarter of its original volume, and
poured into hexane (300 mL). The precipitated solid was filtered
off, washed with hexane, and dried under a vacuum to yield the
product.
[0431] Cis-,
trans-(3R)-methyl-1-(p-carbmethoxyphenyl)-1,2,3,4-tetrahydro-9H-pyrido[3,-
4-b]-indole-3-carboxylate: yield 80%, M.p. 124-125.degree. C.
NMR.sup.1H (.delta., ppm, CDCl.sub.3, 300 MHz): 2.47 (1H, br.s,
NH); 3.05-3.24 (2H, m, Ar--CH.sub.2); 3.68 (3H, s,
CO.sub.2CH.sub.3); 3.79 (3H, s, OCH.sub.3); 3.94 (1H, dd,
ArCH.sub.2CH, J.sub.HH=10.5; 2.8 Hz); 5.27 (0.45H, s, Ar--CH); 5.39
(0.55H, s, Ar--CH) and 7.12-8.18 (9H, m, ArH and indol NH).
NMR.sup.13C (.delta., ppm, CDCl.sub.3, 125.76 MHz): 25.68 (d),
52.84 (dd), 54.80 (s), 56.76 (s), 58.61 (s), 109.16 (d), 111.01
(d), 118.44 (d), 119.76 (d), 122.17 (s), 127.08 (d), 128.62 (d),
130.00 (d), 130.46 (s), 133.60 (d), 136.40 (d), 147.21 (d), 166.66
(d), 173.99 (d). m/z 364 (M.sup.+).
[0432] Cis-,
trans-(3S)-methyl-1-(p-carbmethoxyphenyl)-1,2,3,4-tetrahydro-9H-pyrido[3,-
4-b]-indole-3-carboxylate: yield 60%, M.p. 115-116.degree. C.
NMR.sup.1H (.delta., ppm, CDCl.sub.3, 300 MHz): 2.47 (1H, br.s,
NH); 3.04-3.26 (2H, m, Ar--CH.sub.2); 3.71 (3H, s,
CO.sub.2CH.sub.3); 3.82 (3H, s, OCH.sub.3); 3.96 (1H, dd,
ArCH.sub.2CH, J.sub.HH=10.5; 2.8 Hz); 5.28, 5.39 (1H, twu s,
Ar--CH); 7.14-7.94 (9H, m, ArH and indol NH). NMR.sup.13C (.delta.,
ppm, CDCl.sub.3, 125.76 MHz): 25.70 (d), 52.62 (dd), 54.61 (s),
56.75 (s), 58.41 (s), 109.02 (d), 111.01 (s), 118.32 (d), 119.32
(d), 122.14 (s), 127.03 (d), 128.41 (d), 129.74 (d), 130.23 (d),
133.94 (d), 136.39 (d), 147.24 (d), 166.83 (d), 174.05 (d). % 364
(M.sup.+).
Synthesis of cis-, trans-(3S)- and
(3R)-methyl-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetrahydro--
1H-.beta.-carboline-3-carboxilates
##STR00086##
[0434] Chloroacetyl chloride (0.096 mol) was slowly added to a
cooled-on-ice, stirred solution of 4a or 4b (0.04 mol) and
NaHCO.sub.3 (0.048 mol) in dry chloroform (200 mL). The reaction
mixture was allowed to warm to room temperature and stirred for
four hours. Dichloromethane (200 mL) was added to the reaction
mixture. The resulting solution was washed with NaHCO.sub.3, washed
with a saturated solution of NaCl, dried with MgSO.sub.4, and
concentrated. The solid residue was triturated with ether, filtered
off, and washed with small amount of methanol to yield the
product.
[0435] Cis-,
trans-(3R)-methyl-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetra-
hydro-1H-.beta.-carboline-3-carboxilate: yield 60%, M.p.
273-275.degree. C. NMR.sup.1H (.delta., ppm, DMSO-d6, 500 MHz):
2.86 (3H, s, OCH.sub.3); 3.11-3.50 (2H, m, CH.sub.2Cl); 3.83 (3H,
s, OCH.sub.3); 4.38 (1H, d, ArCH.sub.2, J.sub.HH=14 Hz); 4.77 (1H,
d, ArCH.sub.2, J.sub.HH=14 Hz); 5.19 (1H, d, ArCH, J.sub.HH=2.5
Hz); 6.91 (1H, s, Ar); 7.01-7.08 (2H, dt, Ar, J.sub.HH=25 Hz, 3
Hz); 7.23-7.53 (3H, m, Ar); 7.85 (2H, d, Ar, J.sub.HH=4 Hz); 10.82
(1H, s, NH). NMR.sup.13C (.delta., ppm, DMSO-d6, 125.76 MHz): 21.01
(s), 42.78 (s), 51.09 (s), 51.39 (s), 51.99 (s), 52.48 (s), 106.34
(s), 111.10 (s), 118.02 (s), 118.67 (s), 121.56 (s), 125.79 (s),
128.72 (d), 128.90 (s), 136.36 (s), 144.50 (s), 165.82 (s), 166.87
(s), 169.97 (s). m/z 440 (M.sup.+).
[0436] Cis-,
trans-(3S)-methyl-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetra-
hydro-1H-.beta.-carboline-3-carboxilate: yield 45%, M.p.
272-273.degree. C. NMR.sup.1H (.delta., ppm, DMSO-d6, 500 MHz):
2.84 (1H, s, OCH.sub.3); 3.12-3.49 (2H, m, CH.sub.2Cl); 3.82 (3H,
s, OCH.sub.3); 4.48 (1H, d, ArCH.sub.2, J.sub.HH=14 Hz); 4.86 (1H,
d, ArCH.sub.2, J.sub.HH=14 Hz); 5.23 (1H, d, ArCH, J.sub.HH=2.5
Hz); 6.91 (1H, s, Ar); 7.03-7.10 (2H, dt, Ar, J.sub.HH=25 Hz, 3
Hz); 7.21-7.54 (3H, m, Ar); 7.84 (2H, d, Ar, J.sub.HH=4 Hz); 10.92
(1H, s, NH). NMR.sup.13C (.delta., ppm, DMSO-d6, 125.76 MHz): 21.07
(s), 43.08 (s), 51.15 (s), 51.50 (s), 52.13 (s), 52.53 (s), 106.50
(s), 111.25 (s), 118.10 (s), 118.69 (s), 121.62 (s), 125.87 (s),
128.77 (d), 128.95 (s), 136.43 (s), 144.69 (s), 165.92 (s), 166.97
(s), 170.09 (s). m/z 440 (M.sup.+).
[0437] As will be appreciated by one skilled in the art, other
formula IV compounds (e.g., Compounds 1, 2, and 28-35) may be
synthesized using the above scheme, but starting with a different
aldehyde.
Example 4
[0438] The maximum tolerated dose of Compound 27 was tested in
mice.
[0439] Each mouse in the study was administered a single
intraperitoneal injection of Compound 27 dissolved in sterile PBS
at 100 mg/kg, 200 mg/kg, or 400 mg/kg.
[0440] None of the mice died. Accordingly, the maximum tolerated
dose of Compound 27 was determined to be greater than 400
mg/kg.
Example 5
Analogs of Compound 27
##STR00087## ##STR00088##
[0441] Boc Protection
[0442] Scaffold 2 (SC2) was dissolved in dry CH.sub.3CN (.about.2
mL) along with 3 eq. of triethylamine and stirred under N.sub.2.
Boc anhydride (2+ eq) and DMAP (0.2 eq) were dissolved in 200 uL of
CH.sub.3CN and then added dropwise to the reaction mixture which
changes from yellow to dark brown in color upon addition. The
reaction was heated to 50.degree. C. and left to stir overnight. An
additional 1-3 equivalents of Boc anhydride were added as needed.
Upon reaction completion, CH.sub.3CN was removed under reduced
pressure and the mixture was redissolved in CH.sub.2Cl.sub.2. The
reaction was quenched by addition of 2N NH.sub.4Cl, the organic
layer was removed, washed with brine and dried with
Na.sub.2SO.sub.4. The resulting crude product was purified by
silica chromatography using 2% MeOH in CH.sub.2Cl.sub.2.
Reduction
[0443] Boc-protected product dissolved in a small amount of
CH.sub.2Cl.sub.2 was added to 20-30 mL EtOH. The reaction vessel
was first evacuated and then refilled with Ar several times to
replace the air in the vessel, then a catalytic amount of 10% Pd/C
catalyst was added. The reaction vessel was again evacuated and
then refilled with H.sub.2. The reaction was left to stir
overnight. Product had Rf value 0.21-0.25 in 25% A (A=10% MeOH in
EtOAc) in CH.sub.2Cl.sub.2. Purified crude product by silica gel
chromatography, eluting with a gradient between 10-25% A in
CH.sub.2Cl.sub.2.
Peptide Coupling
[0444] The amine product was dissolved in dry CHCl.sub.3 (.about.1
mL) and stirred under N.sub.2 in a dry conical vial. Biotin-linked
6-aminohexanoic acid (1.1 eq) was dissolved along with EDC (1.2 eq)
and DMAP (0.5 eq) in dry DMF (.about.1 mL). This solution was then
added dropwise to the amine solution and left to stir at 50.degree.
C. overnight. Upon reaction completion, CH.sub.2Cl.sub.2 was added
and then the combined organic layer was washed successively with 1N
HCl, H.sub.2O, saturated aqueous NaHCO.sub.3 and brine, and then
dried with Na.sub.2SO.sub.4. Aqueous layers were also
back-extracted to recover coupled product dissolved in them. The
compound was purified by silica chromatography using 7% MeOH in
CH.sub.2Cl.sub.2.
Deprotection
[0445] The coupled product was dissolved in a solution of 30%
TFA/CH.sub.2Cl.sub.2 and stirred for an hour at room temperature.
The solution turned a reddish pink color. Solvents and TFA were
removed in vacuo, redissolved in CH.sub.2Cl.sub.2 and crude product
was extracted with 5% aqueous K.sub.2CO.sub.3 solution. The organic
phase was washed twice with ddH.sub.2O, dried with Na.sub.2SO.sub.4
and the organic phase concentrated.
Chloroacetylation
[0446] The deprotected product was dissolved in anhydrous
CHCl.sub.3 and 2-3 equivalents of triethylamine were added. To this
was added 0.5 equivalents of chloroacetyl chloride diluted in a
solution of dry CHCl.sub.3. Consecutive half equivalents were added
and the reaction was followed by MS until all the deprotected
product was chloroacetylated.
##STR00089##
Pictet Spengler Reaction
[0447] The hydrochloride salt of L-tryptophan methyl ester was
"free-based" by dissolving it in CH.sub.2Cl.sub.2 and then adding
5% aqueous K.sub.2CO.sub.3. The organic layer was extracted, dried
with Na.sub.2SO.sub.4, and concentrated in vacuo. To a dry vial was
added L-tryptophan methyl ester along with molecular sieves
dissolved in dry CH.sub.2Cl.sub.2. 4-ethynylbenzaldehyde and 0.1 eq
of TFA were added to the reaction mixture at 0.degree. C. and the
solution was then allowed to return to room temperature and stirred
overnight. The following day, 3 equivalents of TFA were added to
the solution and the reaction was allowed to stir for an additional
3-8 hours. Work-up was done by quenching the reaction with
saturated aqueous NaHCO.sub.3, the organic phase was separated,
washed with brine and dried with Na.sub.2SO.sub.4 and then
concentrated to give the crude product. The compound was purified
by dry-loaded silica gel chromatography in 20:80 EtOAc:Hexane with
1% NEt.sub.3 to give the two diastereomers ('cis' Rf=0.29, `trans`
Rf=0.13)
##STR00090##
Chloroacetylation
[0448] The intermediate (one of the diastereomers) was dissolved in
anhydrous CH.sub.2Cl.sub.2 and 2-3 equivalents of triethylamine
added. To this was added 0.5 equivalents of chloroacetyl chloride
diluted in a solution of dry CH.sub.2Cl.sub.2 dropwise at 0.degree.
C. Consecutive half equivalents were added and the reaction was
followed by MS and TLC until all the starting material was
chloroacetylated. Reaction was quenched by addition of saturated
NaHCO.sub.3 and the organic layer was extracted, washed with brine
and dried with Na.sub.2SO.sub.4 and then concentrated. Purification
was done by silica gel chromatography in 20:80 EtOAc:Hexane.
##STR00091##
[0449] Synthesis was analogous to that of para Compound 27 analog,
except methyl 4-formyl-benzoate was used in place 4-ethynyl
benzaldehyde. Purified by silica gel chromatography in EtOAc/Hex
(dry-loading), using gradient from 100% Hexane to 40:60 EtOAc/Hex.
C is diastereomer has Rf 0.35, trans diastereomer has Rf 0.17 in
40:60 EtOAc:Hexane. Instead of chloroacetylation, acryloyl chloride
was used to acryloylate the intermediate in an identical procedure
used for chloroacetylation to give the acrylamide version of
Compound 27.
Synthesis of Ethyl Compound 27 analog
[0450] An analogous procedure to that of the para Compound 27
analog was used, except an L-tryptophan ethyl ester was used and
methyl 4-formyl-benzoate in the pictet-spengler reaction. The two
diastereomers were separated by silica gel chromatography using
EtOAc/Hexane and eluted with a gradient from 100% Hexane to 40:60
EtOAc/Hex. The first diastereomer, D1 has an Rf value of 0.14 and
D2 an Rf value of 0.06 in EtOAc:Hexane. Chloroacetylation was
carried out as described above and the ethyl analog is shown as
follows:
##STR00092##
Synthesis of methyl
(3S)-1-phenyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-carboxylate
(C)
##STR00093##
[0452] To the solution of 0.06 mol of L-tryptophan methyl ester and
0.05 mol of benzaldehyde in 150 ml of benzene, 0.5 ml of
trifluoroacetic acid and 2 g of molecular sieves (4 .ANG.) were
added. The reaction mixture was refluxed for 1 hour, and then 0.138
mol (3 equiv.) of trifluoroacetic acid was added and the mixture
was further refluxed for 3 hours. The reaction mixture was cooled
down to room temperature and diluted with water and basified with
30% solution of NaOH. The organic layer was separated, dried over
MgSO.sub.4 and the solvent was removed in vacuo. The residue was
washed with diethyl ether, and the precipitate thus formed was
filtered off to give the required product (80% yield).
[0453] Melting point: 217-218.degree. C. NMR.sup.1H (.delta., ppm,
CDCl.sub.3, 500 MHz): 2.46 (1H, br.s, NH); 3.04-3.27 (2H, m,
Ar--CH.sub.2); 3.79 (3H, s, OCH.sub.3); 3.97 (1H, dd, ArCH.sub.2CH,
J.sub.HH=10.5; 2.8 Hz); 5.23 (0.75H, s, Ar--CH); 5.39 (0.25H, s,
Ar--CH) and 7.15-7.68 (10H, m, ArH and indol NH).
[0454] NMR.sup.13C (.delta., ppm, CDCl.sub.3, 125.76 MHz): 25.46
(d), 51.73 (d), 54.05 (s), 56.23 (s), 57.77 (s), 106.95 (d), 111.20
(d), 117.60 (d), 118.39 (d), 120.79 (d), 126.52 (s), 127.21 (s),
127.70 (s), 128.13 (s), 135.34 (d), 136.37 (d), 143.02 (d), 173.78
(d). m/z 306 (M.sup.+).
Synthesis of
methyl(3s)-2-(chloroacetyl)-1-phenyl-2,3,4,9-tetrahydro-1H-.beta.-carboli-
ne-3-carboxilates (D)
##STR00094##
[0456] To a stirred, cooled on ice/water mixture containing a
solution of 0.04 mol of compound C and 0.048 mol of NaHCO.sub.3 in
200 ml of dry chloroform, 0.096 mol of chloroacetylchloride was
added drop wise. The reaction mixture was stirred for 4 hours at
room temperature, diluted with dichloromethane and consecutively
washed with a saturated aqueous solution of NaHCO.sub.3 and brine.
The organic layer was separated and dried over MgSO.sub.4. The
solvent was removed on rotary evaporator and the residue was washed
with diethyl ether. The precipitate was filtered off and washed
with a small quantity of methanol to yield the desired product (60%
yield).
[0457] m. p. 173-175.degree. C. NMR.sup.1H (.delta., ppm,
CDCl.sub.3, 500 MHz): 3.00 (2H, br.s, CH.sub.2); 3.22 (1H, m, CH);
3.69 (3H, s, OCH.sub.3); 4.22-4.34 (2H, m, CH.sub.2); 4.93 (1H,
br.s, CH); 6.99 (1H, br.s, Ar); 7.75 (7H, m, Ar); 7.60 (1H, br.s,
Ar); 7.89 (1H, s, NH).
[0458] NMR.sup.13C (.delta., ppm, CDCl.sub.3, 125.76 MHz): 21.62
(s), 42.66 (s), 52.16 (s), 52.22 (s), 53.54 (s), 107.46 (s), 111,
31 (s), 118.57 (d), 119, 85 (d), 122.52 (d), 126.37 (d), 128.26
(s), 129.48 (s), 136.80 (d), 138.96 (d), 167.14 (s), 170.04 (s).
m/z 383.5 (M.sup.+).
Synthesis of
(3S)-1-phenyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-carboxamide
(E)
##STR00095##
[0460] Ammonia gas was bubbled through the solution of 0.01 mol of
compound D in 50 ml of methanol for 2 h at RT, and the reaction
mixture was left for 24 h. The precipitate formed was filtered off
(50% yield) and used in subsequent reactions without further
purification.
[0461] m. p. 153.degree. C. NMR.sup.1H (.delta., ppm, DMSO, 500
MHz): 2.73 (1H, m, CH.sub.2); 2.97 (1H, m, CH.sub.2); 3.20 (1H,
NH); 3.38 (1H, s, CH); 5.22 (1H, s, CH); 7.01 (2H, m, Ar); 7.30
(6H, m, Ar); 7.42 (1H, br.s, Ar); 10.65 (1H, s, NH).
[0462] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 24.89 (s),
51.65 (s), 54.04 (s), 108.01 (s), 110.96 (s), 117.44 (s), 118.16
(s), 120.61 (s), 126.68 (s), 126.87 (s), 127.88 (s), 128.24 (s),
134.21 (s), 136.08 (s), 142.95 (s), 174.91 (s). m/z 291
(M.sup.+).
Synthesis of
(3S)-2-(chloroacetyl)-1-phenyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-c-
arboxamide (F)
##STR00096##
[0464] To the suspension of 0.01 mol of amide E and 0.011 mol of
triethylamine in 50 ml of dry dioxane, 2 equivalents of
chloroacetylchloride was added drop wise at RT. After 1 h at RT the
formed precipitate was filtered off. The filtrate was concentrated
in vacuo, and the obtained residue was washed with diethyl ether.
The precipitate was filtered off and washed with acetonitrile to
give the required product (80% yield).
[0465] m. p. 180-182.degree. C. NMR.sup.1H (.delta., ppm, DMSO, 500
MHz): 3.36 (4H, m, CH.sub.2, CH.sub.2); 4.12 (1H, m, CH); 4.66 (1H,
m, CH); 5.08 (1H, m, NH.sub.2); 6.12 (1H, m, NH.sub.2); 7.25 (9H,
m, Ar); 10.98 (1H, br.s, NH).
[0466] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 24.07 (s),
43.01 (s), 56.41 (s), 56.99 (s), 102.85 (s), 111, 21 (s), 117.73
(s), 118.63 (s), 120.88 (s), 125.82 (s), 126.39 (s), 128.12 (s),
128.98 (s), 134.12 (s), 135.15 (s), 143.95 (s), 167.42 (s), 172.42
(s). m/z 367.5 (M.sup.+).
Synthesis of
2-[(3S)-3-(aminocarbonyl)-1-phenyl-1,3,4,9-tetrahydro-2H-.beta.-carbolin--
2-yl]-2-oxoethyl acetate (G)
##STR00097##
[0468] To the suspension of 0.01 mol of compound D in 20 ml of
DMSO, 2 equivalents of anhydrous sodium acetate was added. The
reaction mixture was stirred for 48 h at RT and diluted with water.
The precipitate was filtered off, dried and washed with diethyl
ether to give the required product (50% yield).
[0469] m. p. 195-197.degree. C. NMR.sup.1H (.delta., ppm, DMSO, 500
MHz): 2.13 (3H, s, CH.sub.3); 2.85 (2H, s, CH.sub.2); 3.36 (5H, m,
CH.sub.2 and OCH.sub.3); 4.85 (1H, br. s, CH); 5.12 (1H, br.s, CH);
6.88-7.55 (9H, m, Ar); 10.94 (1H, s, NH).
[0470] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 21.05 (d),
40.41 (s), 50.95 (d), 51.47 (s), 61.71 (s), 106.16 (s), 111, 19
(s), 118.02 (s), 118.62 (s), 121.47 (s), 125.93 (s), 127.86 (d),
128.69 (s), 129.72 (s), 136.31 (s); 139.53 (s), 166.90 (s), 169.81
(s); 170.25 (s). m/z 406 (M.sup.+).
Synthesis of methyl
(3S)-1-phenyl-2-[(2,3,5,6-tetrafluorophenoxy)acetyl]-2,3,4,9-tetrahydro-1-
H-.beta.-carboline-3-carboxylate (H)
##STR00098##
[0472] Method A. To the solution of 0.01 mol of tetrafluorophenol
in 50 ml of DMFA, 0.013 mol of sodium hydride was added and the
mixture was stirred at RT during 12 h. Then to the reaction mixture
were added 0.01 mol of compound D and 0.01 mol of KI. The resulting
mixture was stirred for 48 h at RT. The reaction mixture was
diluted with water and the precipitate was filtered off. According
to LCMS spectral data, the resulting product had a purity of 76%
and subsequent purification was unsuccessful most likely due to
instability of the final product.
[0473] Method B. Similar to Method A, the reaction mixture was
stirred for 5 days. LC-MS spectrum showed 4% of the product.
Probably decomposition of the product took place as result of
prolonged exposure to alkaline medium.
[0474] Method C. Similar to Method A, but the reaction mixture was
refluxed. LCMS spectrum showed 24% of the product. Attempts to
further purify were unsuccessful. A number of other standard
methods were tried, but they did not lead to a positive result.
Synthesis of methyl
(3S)-1-(1H-imidazol-5-yl)-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-carbox-
ylate (I)
##STR00099##
[0476] To the solution of 0.01 mol of ester A in 50 ml of
trifluoroacetic acid, a solution of 0.011 mol of aldehyde in 10 ml
of methanol was added. The mixture was refluxed for 1 h, cooled
down to RT, and basified to pH 8-9 using a 30% aqueous solution of
NaOH. The precipitate was filtered off and washed with diethyl
ether to give the required product (33% yield).
[0477] m. p. 225-226(BO3r).degree. C. NMR.sup.1H (.delta., ppm,
DMSO, 500 MHz): 2.77 (1H, m, CH.sub.2); 3.00 (1H, m, CH.sub.2);
3.66; 3.71 (3H, s, s, OCH.sub.3); 3.88 (1H, m, CH); 5.27 (1H, s,
CH); 6.95 (3H, m, ArH); 7.28 (1H, br.s., Ar); 7.42 (1H, br.s., Ar);
7.66 (1H, br.s., Ar); 10.40; 10.74 (1H s, s, indol NH).
[0478] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 24.45 (d),
50.61 (s), 51.54 (s), 56.79 (s), 106.08 (s), 111.23 (d), 117.35
(d), 118.28 (d), 120.53 (d), 126.61 (d), 134.91 (s), 135.11 (s),
136.02 (s), 173.04 (d). m/z 296 (M.sup.+).
Synthesis of methyl
(3S)-2-(chloroacetyl)-1-(1H-imidazol-5-yl)-2,3,4,9-tetrahydro-H-.beta.-ca-
rboline-3-carboxylate (J)
##STR00100##
[0480] To the suspension of 0.01 mol of compound I, 0.024 mol of
chloroacetylchloride was added drop wise with stirring at RT. The
reaction mixture was refluxed during 2 h, cooled down, the
precipitated product was filtered off and washed with acetonitrile.
The required product was isolated as HCl salt (60% yield).
[0481] m. p. 250 (decomposition).degree. C. NMR.sup.1H (.delta.,
ppm, dmso, 500 MHz): 3.16 (1H, m, CH.sub.2); 3.30 (3H, s,
OCH.sub.3); 3.55 (3H, m, CH.sub.2 and CH); 4.54 (1H, d, CH.sub.2,
J=14 Hz); 4.90 (1H, d, CH.sub.2, J=14 Hz); 5.13 (1H, br.s, CH);
6.56 (1H, s, lm); 7.03 (1H, br.s, Ar); 7.12 (1H, br.s, Ar); 7.21
(1H, s, lm); 7.33 (1H, br. s, Ar); 7.57, (1H, br. s, Ar); 11.12
(1H, br.s., NH).
[0482] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 21.88 (s),
43.08 (s), 44.31 (s); 52.23 (s), 53.36 (s), 106.53 (s), 111, 39
(s), 118.17 (s), 118.70 (s), 118.80 (s); 121.71 (s), 125.63 (s),
126.81 (s), 131.17 (s), 133.69 (s), 136.61 (s), 167.48 (s), 170.27
(s). m/z 372.5 (M.sup.+).
Synthesis of methyl
(3S)-1-pyridin-4-yl-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-carboxylate
(K)
##STR00101##
[0484] Compound K was synthesized (25% yield) using the same
procedure as for compound I.
[0485] m. p. 220-225 (sublimation).degree. C. NMR.sup.1H (.delta.,
ppm, DMSO, 500 MHz): 3.17 (2H, m, CH.sub.2); 3.63; (3H, s,
OCH.sub.3); 3.77 (1H, br.s, CH); 5.34 (1H, s, CH); 7.04 (2H, br.s,
PyH); 7.30 (4H, m., Ar); 8.05 (2H, br.s., PyH); 10.71 (1H, s, indol
NH).
[0486] NMR.sup.13C (.delta., ppm, DMSO, 125.76 MHz): 24.73 (s),
51.67 (s), 51.95 (s), 53.07 (s), 106.89 (s), 111.13 (s), 117.74
(s), 118.50 (s), 121.08 (s), 123.33 (s), 126.41 (s), 132.92 (s),
136.17s), 149.53 (s), 151.43 (s), 173.65 (s). m/z 307
(M.sup.+).
Synthesis of methyl
(3S)-2-(chloroacetyl)-1-pyridin-4-yl-2,3,4,9-tetrahydro-1H-.beta.-carboli-
ne-3-carboxylate (L)
##STR00102##
[0488] Synthesis was carried out using the same procedure as for
compound J. The product was obtained as a hydrochloride salt with
86% purity (see LCMS spectrum of 90 mg crude sample). During the
purification in polar solvent (CH.sub.3OH, CH.sub.3CN), the
compound decomposed and oxidized into compound M.
##STR00103##
Methyl
(3S)-1-[4-(prop-2-ynyloxy)phenyl]-2,3,4,9-tetrahydro-1H-.beta.-car-
boline-3-carboxylate (O)
##STR00104##
[0490] To a solution of 0.01 mol of ester N in 50 ml of
trifluoroacetic acid a solution of 0.011 mol of aldehyde M in 10 ml
of methanol was added. The reaction mixture was stirred at reflux
for 1 h. 30% aqueous solution of NaOH was added to the cooled to RT
reaction mixture until pH 8-9. The aqueous layer was decanted and
the residue was treated with hexane. The precipitate formed was
filtered off.
[0491] Yield 35%, purity 78% according to LCMS data, m/z 388
(M.sup.+).
Methyl
(3S)-2-(chloroacetyl)-[4-(prop-2-ynyloxy)phenyl]-2,3,4,9-tetrahydro-
-1H-.beta.-carboline-3-carboxylate (Q)
##STR00105##
[0493] To a solution of 0.01 mol of compound P in dry dioxane,
0.024 mol of chloroacetylchloride was added with stirring at RT.
The reaction mixture was refluxed for 1 h, cooled down and the
solvent was removed in vacuo. To the residue, diethyl ether was
added, and the precipitate was filtered off and washed with
methanol. Yield 60%, m. p. 218.degree. C. NMR.sup.1H (.delta., ppm,
dmso, 500 MHz): 2.89 (2H, s, CH.sub.2), 3.21 (5H, m, CH, CH,
OCH.sub.3); 4.36 (1H, d, CH.sub.2, J 12 Hz); 4.77 (1H, d, CH.sub.2,
J 12 Hz); 4.90 (1H, s, CH); 5.22 (1H, br.s, CH); 6.90 (1H, s, Ar);
7.00 (2H, m, Ar); 7.25 (3H, m, Ar); 7.54 (1H, d, Ar, J 7 Hz); 7.88
(1H, d Ar, J 9 Hz); 10.82 (1H, br.s., NH). NMR.sup.13C (.delta.,
ppm, DMSO, 125.76 MHz): 21.10 (s), 44.31 (s); 52.21 (d), 52.49 (d),
77.92 (d), 106.52 (s), 111, 32 (s), 118.16 (s), 118.77 (s), 125.87
(s), 128.34 (s), 129.06 (s), 136.44 (s), 145.08 (s), 164.70 (s),
166.98 (s), 170.08 (s). m/z 463 (M.sup.+).
Methyl(3S)-1-phenyl-2-fluoroacetyl-2,3,4,9-tetrahydro-1H-.beta.-carboline--
3-carboxylate (R)
##STR00106##
[0495] Typical Procedure (the same reaction was performed several
times on different scale):
[0496] Starting material K was prepared according to the previously
described procedure.
[0497] In an oven dried tube, a mixture of K (100 mg, 1 eq.) and
NaHCO.sub.3 (250 mg, 10 eq.) was taken and dry THF (3 mL) was added
under an inert atmosphere. To this mixture was carefully added
fluoroacetyl chloride (0.08 mL, 2 eq., caution: highly toxic and
volatile) and the reaction tube was sealed. The mixture was then
heated at 60.degree. C. overnight. Next day after cooling to RT,
the mixture was quenched with water and diluted with ethyl acetate.
The organic layer was separated, washed with brine solution and
dried (MgSO.sub.4). TLC (60:40, ethyl acetate/hexanes) indicated
slightly higher moving spots (mixture of distereomers) and no
starting material left. There were some low moving impurities.
After concentrating the solution, the resulting solid was dissolved
in methylene chloride and the desired product was obtained after
silica gel column purification using 20 to 40% ethyl acetate/hexane
mixture as eluent. Yield 70 mg (58%, mixture of distereomers).
[0498] MS (367.08, M+H).
[0499] NMR.sup.1H (.delta., ppm, DMSO, 400 MHz): 2.83 (1H, s);
3.01-3.69 (5H, m); 4.76-6.19 (3H, m); 6.83-7.74 (9H, m); 10.91 (1H,
br.s, NH).
[0500] NMR.sup.19F (.delta., ppm, DMSO, 376.33 MHz): -90.90 (s,
first diastereomer), -93.76 (s, second diastereomer).
[0501] HPLC (acetonitrile/water, 20-90% gradient): single peak at
13.4 min
Example 6
Synthesis of the Four Stereoisomers of Compound 27
##STR00107## ##STR00108##
##STR00109## ##STR00110##
[0502] Pictet Spengler Reaction
[0503] To a suspension of 1.2 eq of the corresponding S or R (1
eq.) tryptophan methyl ester hydrochloride (Sigma) in
dichloromethane at room temperature was added 1.3 eq. of
triethylamine (Sigma). The mixture was stirred for 1 hour and then
filtered. The filtrate was concentrated to give the required
product as a clear oil, which was dried under high vacuum for 10
minutes to give the S or R tryptophan methyl ester. Immediately
afterwards, the S or R tryptophan methyl ester along with molecular
sieves were dissolved in dry anhydrous dichloromethane, 1 eq of
methyl 4-formylbenzoate (Sigma) and 0.1 eq of TFA (Sigma) were
added to the reaction mixture and the solution was refluxed for an
hour. After an hour, 3 equivalents of TFA were added to the
solution and the reaction was allowed to stir under reflux
overnight. After cooling to room temperature, a work-up was done by
addition of water, quenching the reaction with 30% NaOH, separating
the organic phase, washing with brine, drying with
Na.sub.2SO.sub.4, and then concentrating to give the crude product.
Each crude product was purified by dry-loaded silica gel
chromatography in a gradient elution from 100% hexane with 1%
NEt.sub.3 to 20:80 EtOAc:Hexane with 1% NEt.sub.3 to give the two
separate diastereomers which are intermediates in the preparation
of Compound 27. Mixed fractions were repurified again with the same
protocol. Yield: 1R,3S Compound 27 Intermediate 18% and 1S,3S
Compound 27 Intermediate 34%; 1R,3R Compound 27 Intermediate 41%
and 1S,3R Compound 27 Intermediate 16%.
Chloroacetylation
[0504] Each of the four intermediates produced above was dissolved
in anhydrous CH.sub.2Cl.sub.2 and 1.1 equivalents of sodium
bicarbonate were added. To this was added 0.5 equivalents of
chloroacetyl chloride dropwise at 0.degree. C. Consecutive half
equivalents were added and the reaction was followed by MS and TLC
until all the starting material was chloroacetylated. The
respective products were filtered and each filtrate was extracted,
washed with brine, dried with Na.sub.2SO.sub.4, and then
concentrated. Each compound was purified by silica gel
chromatography in 20:80 EtOAc:Hexane. Yield: 1R,3S Compound 27
(49%), 1S,3S Compound 27 (5%), 1R,3R Compound 27 (2%), and 1S,3R
Compound 27 (79%).
NMR
[0505] Each of the four stereoisomers of Compound 27 were evaluated
by NMR. FIG. 49 shows that each stereoisomer, 1R,3S Compound 27
(panel A), 1S,3S Compound 27 (panel B), 1S,3R Compound 27 (panel
C), and 1R,3R Compound 27 (panel D) is enantiomerically pure.
Example 7
Synthesis of Stereoisomers of Cyclized Compound 27
##STR00111## ##STR00112##
[0506] Protection of Glycolic Acid
[0507] The protocol according to Nicolaou, K. C., et. al., Studies
toward Diazonamide A: Initial Synthetic Forays Directed toward the
Originally Proposed Structure, 126 (32) JACS 2004, 10162-10173, was
followed. Briefly, 1 eq. of glycolic acid (Sigma) was added to an
oven-dried flask and dried on a vacuum pump for 1 hour, and then
dissolved in anhydrous THF under N.sub.2. To this solution at
0.degree. C. was added a catalytic amount of 4-DMAP (Sigma) and
1.01 eq. NEt.sub.3. Following five minutes of stirring at 0.degree.
C., 1.05 eq. TMSCl (Sigma) was added dropwise, yielding a white
slurry. This suspension was stirred for an additional hour at
0.degree. C., and then mixed for a second hour at room temperature
(RT). A second equivalent (1 eq.) of NEt.sub.3 was added at RT
followed by 1.05 eq. of tert-butyldiphenylsilyl chloride (TBDPSCl)
(Sigma), and this was stirred for 8 hours at 25.degree. C. (or
overnight). Upon completion, the reaction was quenched by the
addition of an aqueous solution of HOAc (about 30% w/v) and stirred
for 1 hour at RT. Water was added, and this solution was extracted
with Et.sub.2O three times. The combined organic layers were then
washed with water twice, dried with Na.sub.2SO.sub.4 and
concentrated in vacuo. The resultant light yellow oil was purified
by flash column chromatography (silica gel, Et.sub.2O/hexanes, 1:1
Rf=0.18). Yield: 78%.
Preparation of Acyl Chloride and Acylation
[0508] 1 eq. of siloprotected glycolic acid from the previous step
was transferred to a reaction flask, concentrated, and dried on a
vacuum pump for one hour. Protected glycolic acid was dissolved in
anhydrous CH.sub.2Cl.sub.2 and 1 eq. of thionyl chloride (Sigma)
was added along with a catalytic amount of anhydrous DMF. Stirring
was allowed for 2 hours under nitrogen. The reaction mixture was
concentrated, redissolved in anhydrous CH.sub.2Cl.sub.2, and
reconcentrated. This process was repeated twice more to remove as
much SOCl.sub.2 as possible. Siloprotected glycolic acid oil was
redissolved in dry dichloromethane, 1 eq. of NEt.sub.3 and 1 eq. of
1S,3S Intermediate (synthesized as described in Example 6) were
added and allowed to stir overnight under nitrogen. The mixture was
dry loaded and purified by flash column chromatography (silica gel,
100% hexane.fwdarw.20:80 ether:hexane, 50:50
ether:hexane.fwdarw.20:80 EtOAc:hexane.fwdarw.100%
EtOAc.fwdarw.100% CHCl.sub.3.fwdarw.5% EtOH/CHCl.sub.3). Yield:
66%.
Deprotection and Cyclization
[0509] 1 eq. of siloprotected 1S,3S Compound 27 was dissolved in
anhydrous dichloromethane in an oven-dried flask under N.sub.2.
TBAF was added in THF (1.0M, Sigma) in an appropriate volume to
give 1.1 eq. of TBAF. Stirring was allowed for five hours. The
reaction was quenched with the addition of saturated NH.sub.4Cl.
The organic layer was extracted three times with dichloromethane.
The organic layers were combined, washed with brine, dried with
Na.sub.2SO.sub.4, filtered and concentrated. 1S,3S Cyclized
Compound 27 was purified by flash column chromatography (silica gel
0.fwdarw.100% EtOAc in hexane). Rf=0.43 in 80:20 EtOAc:Hex.
Yield=17%.
NMR
[0510] The 1S,3S Cyclized Compound 27 obtained according to the
synthesis above and the 1R,3S Cyclized Compound 27 obtained
according to the analogous synthesis as above were each evaluated
by NMR. FIG. 50 shows that 1S,3S Cyclized Compound 27 (panel A) and
1R,3S Cyclized Compound 27 (panel B) are each enantiomerically
pure.
[0511] The synthesis of 1S,3R Cyclized Compound 27 and 1R,3R
Cyclized Compound 27 is analogous to that of 1S,3S Cyclized
Compound 27 and 1R,3S Cyclized Compound 27 as above. It is
considered that 1S,3R Cyclized Compound 27 and 1R,3R Cyclized
Compound 27 obtained from the analogous method to that above are
enantiomerically pure.
Example 8
Biological Testing of the Stereoisomers of Compound 27
[0512] Each of the four stereoisomers of Compound 27 (Example 6)
was tested in four engineered cell lines derived from primary human
fibroblasts. BJ cells were engineered successively to express the
catalytic subunit of human telomerase (hTERT); the SV40 large T and
small T antigens (LT and ST), and an oncogenic allele of HRAS
(HRAS.sup.G12V). These cell lines are identified as BJeH, BJeHLT,
and BJeLR. DRD cells were engineered to express hTERT, oncogenic
HRAS (HRAS.sup.G12V), dominant negative p53, and constitutively
active CDK4/cyclinD, which inactivates the RB protein; the
p53DD/CDK4/cyclinD1 combinations substitute for the SV40 large T
oncoprotein. Of the four cell types, only BJeLR and DRD cells form
tumors in nude mice.
[0513] Cell growth inhibition was determined for the four
stereoisomers of Compound 27, and percent growth inhibition is
plotted in FIG. 51. (1R,3S) Compound 27 (panel A), (1S,3S) Compound
27 panel B), and 1R,3R Compound 27 (panel D) each show no
appreciable growth inhibition. One of the four synthetic
stereoisomers, 1S,3R Compound 27 (panel C), however, showed strong
cell growth inhibition in two cell lines. Moreover, the results
indicate that of the four synthetic stereoisomers, only 1S,3R
Compound 27 has selective lethality towards BJ tumor cell lines
containing mutant HRAS versus isogenic cell lines lacking oncogenic
HRAS.
[0514] The EC.sub.50 of the active stereoisomer, 1S,3R Compound 27,
is about 5 ng/mL (.about.10 nM), whereas the synthesis according to
Example 3 above produces a compound having an EC.sub.50 of about 50
ng/mL (.about.100 nM). It is considered that some racemization
occurred regarding the bond linking the methyl benzoate to the
pyrido-indole group in the synthesis of Example 3. Although a
mixture of diastereomers are produced each having apparent
activity, it has now been discovered that enantiomeric purity does
not result from the Example 3 synthesis. Upon resolving each
stereoisomer, surprisingly it was found that only one of the four
synthetic diastereomers (1S,3R Compound 27) was active in vitro. In
this field, the identification of one out of four stereoisomers
having essentially all of the biological activity was unexpected
and could not have been predicted.
INCORPORATION BY REFERENCE
[0515] All publications and patents referred to herein or listed
below are hereby incorporated by reference in their entirely as if
each individual publication or patent was specifically and
individually indicated to be incorporated by reference. In case of
conflict, the present application, including any definitions
herein, will control.
EQUIVALENTS
[0516] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
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Sequence CWU 1
1
18123DNAArtificialsynthetic construct; VDAC1 F 1cctggacagc
aggaaacagt aac 23220DNAArtificialsynthetic construct; VDAC1 R
2aggcgtcagg gtcaatctga 20320DNAArtificialsynthetic construct; VDAC2
F 3tgattttgct ggacctgcaa 20418DNAArtificialsynthetic construct;
VDAC2 R 4cagcaagcca gccctcat 18520DNAArtificialsynthetic construct;
VDAC3 F 5aatttcgccc tgggttacaa 20621DNAArtificialsynthetic
construct; VDAC3 R 6tcagtgccat cgttcacatg t
21722DNAArtificialsynthetic construct; TfR1 F 7gaaaacagac
agatttgtca tg 22821DNAArtificialsynthetic construct; TfR1 R
8ctcttttgga gatacgtagg g 21921DNAArtificialsynthetic construct;
DMT1 F 9catcactatt atggccctca c 211021DNAArtificialsynthetic
construct; DMT1 R 10gaacatgccc ttgagtacct g
211122DNAArtificialsynthetic construct; HCP1 F 11ggtctttgcc
tttgccacta tc 221223DNAArtificialsynthetic construct; HCP1 R
12caggtgtgat gactaatgac agg 231321DNAArtificialsynthetic construct;
FTH1 F 13cagatcaacc tggagctcta c 211420DNAArtificialsynthetic
construct; FTH1 R 14cttcaaagcc acatcatcgc
201517DNAArtificialsynthetic construct; FTL F 15ggccctggag aaaaagc
171619DNAArtificialsynthetic construct; FTL R 16gaagtgagtc
tccaggaag 191720DNAArtificialsynthetic construct; RPLP0 F
17acgggtacaa acgagtcctg 201820DNAArtificialsynthetic construct;
RPLP0 R 18gccttgacct tttcagcaag 20
* * * * *