U.S. patent application number 14/936949 was filed with the patent office on 2016-02-25 for compounds for use in screening methods for spinal muscular atrophy.
The applicant listed for this patent is The Brigham Women's Hospital, Inc., Indiana University Research and Technology Corporation, The University of Massachusetts. Invention is credited to Elliot Androphy, Jonathan Cherry, Gregory D. Cuny, Marcie A. Glicksman.
Application Number | 20160052935 14/936949 |
Document ID | / |
Family ID | 49916705 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160052935 |
Kind Code |
A1 |
Androphy; Elliot ; et
al. |
February 25, 2016 |
COMPOUNDS FOR USE IN SCREENING METHODS FOR SPINAL MUSCULAR
ATROPHY
Abstract
Disclosed herein are compositions and methods for treatment of
spinal muscular atrophy (SMA). In certain embodiments, compounds
are provided that increase full-length survival of motor neuron
(SMN) protein production by an SMN2 gene.
Inventors: |
Androphy; Elliot;
(Indianapolis, IN) ; Cuny; Gregory D.; (Houston,
TX) ; Cherry; Jonathan; (Carmel, IN) ;
Glicksman; Marcie A.; (Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Indiana University Research and Technology Corporation
The Brigham Women's Hospital, Inc.
The University of Massachusetts |
Indianapolis
Boston
Boston |
IN
MA
MA |
US
US
US |
|
|
Family ID: |
49916705 |
Appl. No.: |
14/936949 |
Filed: |
November 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13941099 |
Jul 12, 2013 |
9212209 |
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14936949 |
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61671425 |
Jul 13, 2012 |
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Current U.S.
Class: |
514/211.05 ;
435/369; 435/375; 514/255.05; 514/272; 514/300; 514/313; 514/340;
514/363; 514/371; 514/377; 514/378; 514/380; 514/398; 514/406;
540/490; 544/321; 544/405; 546/121; 546/160; 546/272.1; 548/139;
548/195; 548/233; 548/246; 548/248; 548/332.5; 548/374.1 |
Current CPC
Class: |
C07D 213/75 20130101;
C07D 261/06 20130101; C07D 277/46 20130101; C07D 261/18 20130101;
C07D 471/04 20130101; C07D 215/44 20130101; C07D 215/227 20130101;
C07D 239/47 20130101; C07D 239/82 20130101; C07D 263/48 20130101;
C07D 231/14 20130101; C07D 267/14 20130101; C07D 417/04 20130101;
C07D 233/88 20130101; C07C 2603/20 20170501; C07D 261/14 20130101;
C07C 2601/08 20170501; C07D 285/135 20130101; C07D 413/12 20130101;
C07D 405/04 20130101; C07C 311/37 20130101; C07D 405/12 20130101;
C07D 493/04 20130101; C07K 14/47 20130101; C07D 401/04 20130101;
C07D 487/04 20130101; C07D 221/10 20130101; C07D 417/12 20130101;
A61P 21/00 20180101 |
International
Class: |
C07D 493/04 20060101
C07D493/04; C07D 413/12 20060101 C07D413/12; C07D 261/14 20060101
C07D261/14; C07D 231/14 20060101 C07D231/14; C07D 285/135 20060101
C07D285/135; C07D 277/46 20060101 C07D277/46; C07D 263/48 20060101
C07D263/48; C07D 233/88 20060101 C07D233/88; C07D 239/47 20060101
C07D239/47; C07D 417/12 20060101 C07D417/12; C07D 215/44 20060101
C07D215/44; C07D 471/04 20060101 C07D471/04; C07D 417/04 20060101
C07D417/04; C07D 215/227 20060101 C07D215/227; C07D 487/04 20060101
C07D487/04; C07D 221/10 20060101 C07D221/10; C07D 405/04 20060101
C07D405/04; C07D 401/04 20060101 C07D401/04; C07D 239/82 20060101
C07D239/82; C07D 267/14 20060101 C07D267/14; C07D 261/18 20060101
C07D261/18 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers HD064850 and NS064349 awarded by the National Institutes of
Health. The U.S. Government has certain rights in the invention.
Claims
1. A compound of formula I: ##STR00320## or a pharmaceutically
acceptable salt thereof, wherein each of Ring A and Ring B is
independently an optionally substituted group selected from phenyl,
a 3-7 membered saturated or partially unsaturated carbocyclic ring,
an 8-10 membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 3-7 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic
saturated or partially unsaturated heterocyclic ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur; L.sup.1 is independently a covalent bond or an optionally
substituted bivalent C.sub.1-6 hydrocarbon chain, wherein one or
more methylene units of L.sup.1 are optionally and independently
replaced by --O--, --S--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R)--, S--(O)--, --S(O).sub.2--, --S(O).sub.2N(R)--,
--N(R)S(O).sub.2--, --OC(O)--, or --(O)0-; each R' is independently
--R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: two R' on the same
nitrogen are taken together with their intervening atoms to form a
3-7 membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur; and each R is hydrogen,
or an optionally substituted group selected from C1-6 aliphatic,
phenyl, a 3-7 membered saturated or partially unsaturated
carbocyclic ring, an 8-10 membered bicyclic saturated, partially
unsaturated or aryl carbocyclic ring, a 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, an 8-10 membered
bicyclic saturated or partially unsaturated heterocyclic ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
2. The compound according to claim 1, wherein said compound has the
structure of formula II: ##STR00321## or a pharmaceutically
acceptable salt thereof, wherein each of Ring A' and Ring B' is
independently an optionally substituted group selected from phenyl,
an 8-10 membered bicyclic aryl ring, a 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur; L.sup.2 is --C(O)N(R')--; each R' is
independently --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: two R'
on the same nitrogen are taken together with their intervening
atoms to form a 3-7 membered heterocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur; and each R is hydrogen, or an optionally substituted group
selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered saturated
or partially unsaturated carbocyclic ring, an 8-10 membered
bicyclic saturated, partially unsaturated or aryl carbocyclic ring,
a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
3. The compound according to claim 2, wherein said compound has the
structure of formula 11-a: ##STR00322## or a pharmaceutically
acceptable salt thereof, wherein: Ring A'' is an optionally
substituted phenyl or benzimidazolyl ring; Ring B'' is an
optionally substituted 5-6 membered heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur; L.sup.3 is --C(O)NH--; each R.sup.4 is independently
halogen or R; each R.sup.5 is independently an optionally
substituted C.sub.1-6 aliphatic; each of d and e is independently
0-5; and each R is hydrogen, or an optionally substituted group
selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered saturated
or partially unsaturated carbocyclic ring, an 8-10 membered
bicyclic saturated, partially unsaturated or aryl carbocyclic ring,
a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
4. The compound according to claim 3, wherein said compound has the
structure of: ##STR00323##
5. The compound according to claim 3, wherein said compound has the
structure of: ##STR00324##
6. The compound according to claim 3, wherein said compound has the
structure of: ##STR00325##
7. The compound according to claim 3, wherein said compound has the
structure of: ##STR00326##
8. The compound according to claim 1, wherein said compound has the
structure of formula III: ##STR00327## or a pharmaceutically
acceptable salt thereof, wherein Ring C'' is independently an
optionally substituted group selected from phenyl, an 8-10 membered
bicyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; each of R.sup.1, R.sup.2 and R.sup.3 is
independently halogen, R', --C(O)R', --C(S)R', --CO.sub.2R',
--C(O)N(R').sub.2, --C(S)N(R').sub.2, --S(O)R', --SO.sub.2R',
--SO.sub.2N(R').sub.2, --OR', --O--(C.sub.1-6
aliphatic)-N(R').sub.2, --O0-(C.sub.1-6 aliphatic)-OR', --OC(O)R',
--SR', --NO.sub.2, --N(R').sub.2, --NR'C(O)R', --NR'C(O)OR',
--NR'C(O)N(R').sub.2, --NR'SO.sub.2R', --NR'SO.sub.2N(R').sub.2, or
--NR'OR'; a is 1-4; b is 1-5; X.sup.1 is --C(R.sup.x).sub.2--,
--NR.sup.x--, --NR.sup.xC(R.sup.x).sub.2-- or
--OC(R.sup.x).sub.2--; X.sup.2 is --C(R.sup.x).sub.2-- or
--NR.sup.x--; each R.sup.x is independently R', --(C.sub.1-6
aliphatic)-N(R).sub.2, or --(C.sub.1-6 aliphatic)-OR'; each R' is
independently --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: two R'
on the same nitrogen are taken together with their intervening
atoms to form a 3-7 membered heterocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur; and each R is hydrogen, or an optionally substituted group
selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered saturated
or partially unsaturated carbocyclic ring, an 8-10 membered
bicyclic saturated, partially unsaturated or aryl carbocyclic ring,
a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
9. The compound according to claim 1, selected from those depicted
in Table 1, or a pharmaceutically acceptable salt thereof.
10. A composition comprising a compound according to claim 1 and a
pharmaceutically acceptable carrier, adjuvant or vehicle.
11. A method for increasing the production of full-length SMN
protein in a cell having an SMN2 gene, the method comprising
contacting the cell with a compound or composition according to
claim 1.
12. The method of claim 11, wherein the full-length SMN protein is
produced by upregulation of SMN2 activity.
13. The method of claim 12, wherein the upregulation of SMN2
activity includes increased expression of full-length SMN2
transcripts.
14. A method of increasing the production of full-length SMN
protein in a cell having an SMN2 gene, the method comprising
contacting the cell with a compound or composition according to
claim 2.
15. The method of claim 14, wherein the full-length SMN protein is
produced by upregulation of SMN2 activity.
16. A method of increasing the production of full-length SMN
protein in a cell having an SMN2 gene, the method comprising
contacting the cell with a compound or composition according to
claim 3.
17. The method of claim 16, wherein the full-length SMN protein is
produced by upregulation of SMN2 activity.
18. A method of treating a patient susceptible to or having spinal
muscular atrophy, the method comprising administering to the
patient a therapeutically effective amount of a compound or
composition of claim 1.
19. A method of treating a patient susceptible to or having spinal
muscular atrophy, the method comprising administering to the
patient a therapeutically effective amount of a compound or
composition of claim 2.
20. A method of treating a patient susceptible to or having spinal
muscular atrophy, the method comprising administering to the
patient a therapeutically effective amount of a compound or
composition of claim 3.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
Non-provisional application Ser. No. 13/941,099 filed Jul. 12,
2013, which claims priority to U.S. Provisional Patent Application
No. 61/671,425 filed on Jul. 13, 2012, both of which are hereby
incorporated by reference in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0003] A paper copy of the Sequence Listing and a computer readable
form of the sequence containing the file named "31377-18 (IURTC
12111)_ST25.txt", which is 54,433 bytes in size (as measured in
MS-DOS), are provided herein and are herein incorporated by
reference. This Sequence Listing consists of SEQ ID NOS: 1-9.
FIELD OF DISCLOSURE
[0004] The present disclosure relates to pharmaceutically active
compounds useful for treating, or lessening the severity of, spinal
muscular atrophy.
BACKGROUND OF DISCLOSURE
[0005] Spinal muscular atrophy (SMA) is a neurological disorder
that results from loss of function of the anterior horn cells in
the spinal cord, manifesting as progressive motor weakness, muscle
wasting, and paralysis. SMA is caused by insufficient levels of the
survival motor neuron (SMN) protein. The SMN locus on chromosome
5q13 contains two inverted copies of SMN called SMN1 and SMN2. Most
cases of SMA harbor homozygous deletions of the SMN1 gene and
retain at least one copy of SMN2. With a carrier rate of about 1 in
40, SMA is estimated to be the most frequent genetic cause of
infant mortality.
[0006] SMN2 is a gene duplication of SMN1 with the same predicted
amino acid coding capacity. The nucleotide sequences of SMN1 and
SMN2 are nearly identical. A critical difference is a C to T
transition at the +6 position in exon 7, which dramatically
influences the splicing pattern in these genes. Greater than 90% of
SMN1 transcripts include exon 7, while there is less than 15% exon
7 inclusion in SMN2 transcripts. This alternatively spliced product
produces a truncated and unstable form of the SMN protein. Any
increase in the inclusion of exon 7 in SMN2 transcripts would
result in higher levels of full length SMN protein. A treatment
that increases the amount of full length SMN2 mRNA should result in
increased levels of SMN protein. Based on this premise, an in vivo
screen that can detect increases in full-length exon 7 included
SMN2 transcripts was developed.
[0007] A splicing reporter that fused SMN exons 6, 7, and 8 and
their introns in frame with firefly luciferase and was expressed
from a CMV promoter in C33a cells has previously been constructed.
It was found that this reporter could recapitulate changes in
splicing observed with over-expression of the splicing factor
Tra2.beta.. This assay was used successfully to identify compounds
that increase the amount of full-length transcript produced by the
SMN2 gene and SMN protein in fibroblasts isolated from an SMA
patient. Another study used a SMN2 promoter based reporter to
screen a library of small molecules for ability to increase SMN
expression levels in NSC34 cells. This reporter measured only SMN2
specific transcription and lacked any SMN gene sequence.
[0008] It has been reported that histone deacetylase (HDAC)
inhibitors such as sodium butyrate, trichostatin A (TSA), valproic
acid, suberoylanilide hydroxamic acid (SAHA) and LBH589 increase
SMN transcription and inclusion of exon 7. For many of these HDACs,
relatively high (micromolar or millimolar) concentrations of these
compounds are necessary. These activators are non-specific and will
alter transcription of many genes so long-term safety has been
questioned. However, type I severe SMA is fatal and short-term
administration of such compounds may provide limited benefits.
[0009] A first generation splicing assay had low signal intensity,
high basal expression of SMN-luciferase, and became less responsive
with serial cell passage. These cells were determined to be
unsuitable for high-throughput screening (HTS). The reporter system
was redesigned and a more stable and reproducible assay has now
been built that may be used for HTS.
[0010] Particularly, described herein is a clonal second generation
SMN-luciferase reporter cell line that combines the strengths of
both the promoter-based assay and a previous splicing reporter.
This assay is much more robust, has lower well-to-well variation,
and displays more stable luciferase expression that does not change
with serial passage. It also faithfully reproduces the reported
activity of an array of drug-like compounds that have been shown to
increase SMN expression levels. This reporter can detect changes in
SMN2 levels in response to overexpression of splicing factors such
as Tra2.beta.. This assay is a vast improvement on the previous
generation of reporters and represents a valuable tool for further
identification and characterization of compounds that increase
expression of full-length SMN protein from the SMN2 gene.
[0011] Additionally, there is a need for new drugs to treat spinal
muscular atrophy. SMN reporters can be used as tools for
identifying and characterizing protein factors and chemical
compounds that increase expression of full-length SMN2 transcripts.
Results from HTSs to identify novel compounds that increase SMN2
expression using this cell based SMN-luciferase reporter assay are
also described herein.
[0012] As described herein, the present disclosure provides
compounds useful for treating or lessening the severity of spinal
muscular atrophy. The present disclosure also provides methods of
treating or lessening the severity of spinal muscular atrophy
comprising administering to a patient susceptible to or having
spinal muscular atrophy a compound or composition of the present
disclosure.
BRIEF DESCRIPTION
[0013] The present disclosure is generally related to compounds and
methods for treatment of spinal muscular atrophy (SMA) utilizing
the compounds. In certain embodiments, compounds are provided that
increase full-length survival of motor neuron (SMN) protein
production by an SMN2 gene.
[0014] Accordingly, in one aspect, the present disclosure is
directed to a compound of formula I:
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein [0015] each
of Ring A and Ring B is independently an optionally substituted
group selected from phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; [0016] L.sup.1 is independently a covalent bond
or an optionally substituted bivalent C1-6 hydrocarbon chain,
wherein one or more methylene units of L.sup.1 are optionally and
independently replaced by --O--, --S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--OC(O)--, or --C(O)O--; [0017] each R' is independently --R,
--C(O)R, --CO2R, or --SO2R, or: [0018] two R' on the same nitrogen
are taken together with their intervening atoms to form a 3-7
membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur; and [0019] each R is
hydrogen, or an optionally substituted group selected from C1-6
aliphatic, phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0020] In another aspect, the present disclosure is directed to a
composition comprising a compound and a pharmaceutically acceptable
carrier, adjuvant or vehicle. The compound has the formula of
formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein [0021] each
of Ring A and Ring B is independently an optionally substituted
group selected from phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; [0022] L.sup.1 is independently a covalent bond
or an optionally substituted bivalent C1-6 hydrocarbon chain,
wherein one or more methylene units of L.sup.1 are optionally and
independently replaced by --O--, --S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--OC(O)--, or --C(O)O--; [0023] each R' is independently --R,
--C(O)R, --CO2R, or --SO2R, or: [0024] two R' on the same nitrogen
are taken together with their intervening atoms to form a 3-7
membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur; and [0025] each R is
hydrogen, or an optionally substituted group selected from C1-6
aliphatic, phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0026] In yet another aspect, the present disclosure is directed to
a method for increasing the production of full-length SMN protein
in a cell having an SMN2 gene. The method comprises contacting the
cell with a compound or composition. In some embodiments, the
compound has the formula of formula I:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein [0027] each
of Ring A and Ring B is independently an optionally substituted
group selected from phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; [0028] L.sup.1 is independently a covalent bond
or an optionally substituted bivalent C1-6 hydrocarbon chain,
wherein one or more methylene units of L.sup.1 are optionally and
independently replaced by --O--, --S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--OC(O)--, or --C(O)O--; [0029] each R' is independently --R,
--C(O)R, --CO2R, or --SO2R, or: [0030] two R' on the same nitrogen
are taken together with their intervening atoms to form a 3-7
membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur; and [0031] each R is
hydrogen, or an optionally substituted group selected from C1-6
aliphatic, phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In other embodiments, a composition is
administered, the composition includes the compound described above
and a pharmaceutically acceptable carrier, adjuvant or vehicle.
[0032] In some embodiments, the full-length SMN protein is produced
by upregulation of SMN2 activity. In one embodiment, the
upregulation of SMN2 activity includes increased expression of
full-length SMN2 transcripts.
[0033] In another aspect, the present disclosure is directed to a
method of treating a patient susceptible to or having spinal
muscular atrophy. The method comprises administering to the patient
a therapeutically effective amount of the compound or composition
described above.
[0034] In another aspect, the present disclosure is directed to a
vector. The vector comprises a survival motor neuron (SMN) promoter
selected from the group consisting of a survival motor neuron 1
(SMN1) promoter and a survival motor neuron 2 (SMN2) promoter; a
transcription start site; a nucleic acid encoding exons 1-8 of the
SMN gene and encoding introns 6-8 of the SMN gene; and a reporter
gene. In yet another aspect, the present disclosure is directed to
a host cell comprising the vector.
[0035] In another aspect, the present disclosure is directed to a
vector. The vector comprises a survival motor neuron 1 (SMN1)
promoter; a transcription start site; a nucleic acid encoding exons
1-8 of the SMN1 gene and encoding introns 6-8 of the SMN1 gene; and
a reporter gene. In yet another aspect, the present disclosure is
directed to a host cell comprising the vector.
[0036] In yet another aspect, the present disclosure is directed to
a vector. The vector comprises a survival motor neuron 2 (SMN2)
promoter; a transcription start site; a nucleic acid encoding exons
1-8 of the SMN2 gene and encoding introns 6-8 of the SMN2 gene; and
a reporter gene. In yet another aspect, the present disclosure is
directed to a host cell comprising the vector.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIGS. 1A-1D show SMN-reporter mini-genes. FIGS. 1A and 1B
illustrate the changes made to the new reporter (see text). The
asterisk denotes a restriction site included during cloning that is
unique to the SMN reporter gene. These reporters produce a
SMN-luciferase fusion protein when exon 7 is included (FIG. 1C) but
not when exon 7 is skipped (FIG. 1D). .about.90% of mRNA from the
SMN2-reporter mini-gene skips exon 7.
[0038] FIGS. 2A-2D show reporter assay validation. FIG. 2A
illustrates a comparison of luciferase activity for equivalent
numbers of mixed population SMN1-luc, mixed population SMN2-luc,
clonal SMN1-luc and clonal SMN2-luc cells. Luciferase activity,
scored as relative light units (RLU). FIG. 2B illustrates the
detection of SMN-luciferase fusion protein in HEK parent, SMN1-luc,
and SMN2-luc reporter cells. Lysates were blotted for the fusion
protein with an anti-luciferase antibody and endogenous SMN and
compared to actin and tubulin. FIG. 2C illustrates the end point
RT-PCR used to compare mRNA species of SMN1-luc and SMN2-luc cells.
Primer pairs were designed to amplify both full-length and exon 7
excluded SMN-luciferase mRNA. Percent inclusion was determined by
comparing band intensities using QUANTITY ONE software. FIG. 2D
illustrates that SMN-luciferase fusion stability was determined by
treating cells with 10 .mu.M cycloheximide and measuring luciferase
activity at various time points. Data were plotted using PRISM4
(Graphpad Software Inc.) and non-linear regression was used to
determine protein half-life.
[0039] FIGS. 3A-3E show activity of known compounds in the reporter
cells. FIG. 3A depicts results of sodium butyrate, SAHA, TSA,
aclarubicin, indoprofen, valproic acid, and tobramycin tested in
6-point dose response experiments. Each compound was tested at
concentrations previously reported to increase SMN protein levels.
Black square with solid line SMN2-luc cells; gray
triangle--SMN1-luc cells; black circle with dotted line--SV40
control. Y-axis represents % activation relative to DMSO control.
All points were tested in quadruplicate and plotted as mean.+-.SEM.
Curves were created by linear regression using PRISM4 (Graphpad
Software Inc.). FIGS. 3B and 3C illustrate SMN1-luc cells treated
for 24 hours at increasing concentrations of SAHA. The amount of
SMN-luciferase fusion protein detected by anti-luciferase antibody
(FIG. 3B) is similar to the % increase in luciferase activity in
the same samples (FIG. 3C). FIGS. 3D and 3E illustrate SMN2-luc
cells treated for 24 hours with increasing amounts of SAHA or
sodium butyrate. The increase in SMN-luciferase fusion protein
(FIG. 3D) correlates with luciferase activity (FIG. 3E).
Experiments were performed 3 times with similar results. Blots
shown are representative.
[0040] FIGS. 4A and 4B show analysis of SMN-luciferase fusion
transcripts by qRT-PCR. FIG. 4A is a schematic of the primer design
for qRT-PCR. Primer pairs were chosen to amplify only the
SMN-luciferase fusion transcripts but not endogenous SMN. Primer 1
overlaps a unique Xho I site (*). Primers 1 and 2 can only amplify
full-length SMN-luc transcripts that contain exon 7. Primers 1 and
3 amplify SMN-luc reporter transcripts (both exon 7 included and
excluded). FIG. 4B illustrates the comparison of increases in
amount of total reporter transcripts (lined bars) and amount of
exon 7 included reporter transcripts (white bars). Cells were
treated at increasing concentrations of compound for 24 hours.
Percent increase was calculated in relation to treatment with DMSO
and normalized to GAPDH.
[0041] FIGS. 5A-5C show over-expression of the splicing factors
hTra2.beta. and SF2/ASF. SMN2-luciferase reporter cells were
transfected with increasing amounts of HA-tagged hTra2.beta. or
SF2/ASF. Cells were incubated for 48 hours and assayed for
luciferase activity. FIG. 5A shows the percent increase calculated
in relation to treatment with control DNA transfection and
normalized to internal renilla control. FIG. 5B illustrates that
transfected protein expression was confirmed using anti-HA
antibody. The asterisk denotes a background HA band. Actin was used
as a loading control. FIG. 5C illustrates the comparison of
increases in amount of total reporter transcripts (gray bars), and
amount of exon 7 included reporter transcripts (white bars).
Percent increase was calculated in relation to treatment with DMSO
and normalized to GAPDH.
[0042] FIGS. 6A-6C show hit confirmation in the reporter cells.
FIG. 6A depicts structures of three hits from the high-throughput
screen. FIG. 6B illustrates 12-point dose response experiments.
Each compound was tested with the reporter cell lines at 12
concentrations (0.17, 0.5, 1.5, 4.5, 13.7, 41, 123, 370, 1111,
3333, 10,000, 30,000 nM) Black-SMN2-luc cells; grey--SMN1-luc
cells; dotted line--SV40 control. Y-axis represents % activation
over DMSO control. All points were tested in quadruplicate and
plotted as mean.+-.SEM. Curves were created by linear regression
using Prism4 (GraphPad Software Inc.). FIG. 6C illustrates primary
human fibroblast lysates from carrier (3814; SMN1.sup.+/-;
SMN2.sup.+/+) and depicts SMA (3813; SMN1.sup.-/-; SMN2.sup.+/+)
cells that were blotted with antibodies to SMN and a-tubulin. Cells
were treated for 48 hours with increasing concentrations of
compound. Fold increase was calculated in relation to DMSO treated
3813 and normalized to tubulin levels. Experiments were performed 3
times. Blots shown are representative.
[0043] FIG. 7 shows original and new reporter assay comparison.
Comparison of luciferase activity for equivalent numbers of
previous generation reporter cells, mixed population SMN1-luc,
mixed population SMN2-luc, clonal SMN1-luc and clonal SMN2-luc
cells. Luciferase activity was scored as relative light units
(RLU).
[0044] FIG. 8 shows survival proportions of animals in a mouse
model of spinal muscular atrophy treated with compound 76070, DMSO,
or untreated.
[0045] FIG. 9 shows average weights of animals in a mouse model of
spinal muscular atrophy treated with compound 76070, DMSO, or
untreated.
[0046] FIG. 10 shows percent weight gained from birth to peak of
animals in a mouse model of spinal muscular atrophy treated with
compound 76070, DMSO, or untreated.
[0047] FIG. 11 shows actual time to right of the compound
76070-treated group. Animals are placed on their backs and assayed
for the time it requires for them to right themselves. Thirty (30)
seconds or greater is considered a failure to right and scored as
30 sec.
[0048] FIG. 12 shows actual time to right of the DMSO-treated
group. Animals are placed on their backs and assayed for the time
it requires for them to right themselves. Thirty (30) seconds or
greater is considered a failure to right and scored as 30 sec.
[0049] FIG. 13 shows actual time to right of the untreated group.
Animals are placed on their backs and assayed for the time it
requires for them to right themselves. Thirty (30) seconds or
greater is considered a failure to right and scored as 30 sec.
[0050] FIG. 14 shows the average time to right between postnatal
day 7 and postnatal day 20 of the untreated, DMSO, and compound
76070-treated groups. Animals are placed on their backs and assayed
for the time it requires for them to right themselves. Thirty (30)
seconds or greater is considered a failure to right and scored as
30 sec.
[0051] FIG. 15 shows the percentage of animals able to right
between postnatal day 7 and postnatal day 20 of the untreated,
DMSO, and compound 76070-treated groups.
[0052] FIG. 16 shows the actual time to right on postnatal day 9 of
untreated, DMSO, and compound 76070-treated groups.
[0053] FIG. 17 shows survival proportions of animals in a mouse
model of spinal muscular atrophy treated with compound 212014,
DMSO, or untreated.
[0054] FIG. 18 shows average weights of animals in a mouse model of
spinal muscular atrophy treated with compound 212014, DMSO, or
untreated.
[0055] FIG. 19 shows percent weight gained from birth to peak of
animals in a mouse model of spinal muscular atrophy treated with
compound 212014, DMSO, or untreated.
[0056] FIG. 20 shows actual time to right of the compound
212014-treated group.
[0057] FIG. 21 shows actual time to right of the untreated
group.
[0058] FIG. 22 shows actual time to right of the DMSO-treated
group.
[0059] FIG. 23 shows percentage of animals able to right between
postnatal day 7 and postnatal day 19 of the untreated, DMSO, and
compound 212014-treated groups.
[0060] FIG. 24 shows the average time to right between postnatal
day 7 and postnatal day 21 of the untreated, DMSO, and compound
212014-treated groups.
[0061] FIG. 25A shows luciferase activities (-.quadrature.-) in
SMN2 reporter cells treated with LDDN-75654, LDN-212016, LDN-76070,
and LDN-212391. The curve represented by -.DELTA. is the internal
control renilla luciferase and the curve represented by
-.smallcircle.- is the luciferase activity in SMN1 reporter
cells.
[0062] FIG. 25B shows SMN protein levels in 3813 cells by
immunoblot and gem counts. Cells were treated with LDN-75654 (left
two panels) or LDN-76070 (right two panels) for 48 hours. Graphs
plot the number of gems per 100 nuclei where 3813 cells are
designated by lined bars and 3814 by solid bars. Data are presented
as mean.+-.SEM for three experiments and were analyzed using Prism4
(GraphPad Software Inc.). Each data point was compared to DMSO
control using t-test. * (P<0.05) ** (P<0.01) ***
(P<0.001).
[0063] FIG. 26A shows survival proportions of animals in a mouse
model of spinal muscular atrophy treated with compounds. FIG. 26B
shows average weights of animals in a mouse model of spinal
muscular atrophy treated with compounds.
[0064] FIG. 27 shows survival proportions of animals in a mouse
model of spinal muscular atrophy treated with compounds. Animals
were treated once a day with 20 mg/kg by IP injection starting on
post natal day (PND) 1.
[0065] FIG. 28 shows time to right of mice treated with compounds.
Animals are placed on their backs and assayed for the time it
requires for them to right themselves. Thirty (30) seconds or
greater is considered a failure to right and scored as 30 sec.
[0066] FIG. 29 shows SMN protein levels in treated animals. Animals
were harvested on postnatal day 7 and blotted for SMN and IP90.
[0067] FIG. 30 shows a vector map of the SMN1 luciferase vector
(SEQ ID NO:8).
[0068] FIG. 31 shows a vector map of the SMN2 luniferase vector
(SEQ ID NO:9).
DETAILED DESCRIPTION
1. General Description of Compounds of the Disclosure
[0069] According to one embodiment, the present disclosure provides
a compound of formula I:
##STR00004##
or a pharmaceutically acceptable salt thereof, wherein: [0070] each
of Ring A and Ring B is independently an optionally substituted
group selected from phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; [0071] L.sup.1 is independently a covalent bond
or an optionally substituted bivalent C.sub.1-6 hydrocarbon chain,
wherein one or more methylene units of L.sup.1 are optionally and
independently replaced by --O--, --S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--, --S(O)2-,
--S(O)2N(R')--, --N(R')S(O)2-, --OC(O)--, or --C(O)O--; [0072] each
R' is independently --R, --C(O)R, --CO2R, or --SO2R, or: [0073] two
R' on the same nitrogen are taken together with their intervening
atoms to form a 3-7 membered heterocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur; and [0074] each R is hydrogen, or an optionally substituted
group selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered
saturated or partially unsaturated carbocyclic ring, an 8-10
membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 3-7 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic
saturated or partially unsaturated heterocyclic ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0075] According to one embodiment, the present disclosure provides
a compound of formula II:
##STR00005##
or a pharmaceutically acceptable salt thereof, wherein: [0076] each
of Ring A' and Ring B' is independently an optionally substituted
group selected from phenyl, an 8-10 membered bicyclic aryl ring, a
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, or an 8-10
membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; [0077]
L.sup.2 is --C(O)N(R')--; [0078] R' is --R, --C(O)R, --CO.sub.2R,
or --SO.sub.2R, or: [0079] two R' on the same nitrogen are taken
together with their intervening atoms to form a 3-7 membered
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, and sulfur; and [0080] each R is hydrogen,
or an optionally substituted group selected from C.sub.1-6
aliphatic, phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0081] According to one embodiment, the present disclosure provides
a compound of formula III:
##STR00006##
or a pharmaceutically acceptable salt thereof, wherein: [0082] Ring
C'' is independently an optionally substituted group selected from
phenyl, an 8-10 membered bicyclic aryl ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur; [0083] each of R.sup.1,
R.sup.2 and R.sup.3 is independently halogen, R', --C(O)R',
--C(S)R', --CO2R', --C(O)N(R')2, --C(S)N(R').sub.2, --S(O)R',
--SO.sub.2R', --SO2N(R').sub.2, --OR', --O--(C.sub.1-6
aliphatic)-N(R').sub.2, --O--(C.sub.1-6 aliphatic)-OR', --OC(O)R',
--SR', --NO.sub.2, --N(R').sub.2, --NR'C(O)R', --NR'C(O)OR',
--NR'C(O)N(R').sub.2, --NR'SO.sub.2R', --NR'SO.sub.2N(R').sub.2, or
--NR'OR'; [0084] a is 1-4; [0085] b is 1-5; [0086] X.sup.1 is
--C(R.sup.x).sub.2--, --NR.sup.x--, --NR.sup.xC(R.sup.x).sub.2-- or
--OC(R.sup.x).sub.2--; X.sup.2 is --C(R.sup.x).sub.2-- or
--NR.sup.x--; [0087] each R.sup.x is R', --(C1-6
aliphatic)-N(R').sub.2, or --(C.sub.1-6 aliphatic)-OR'; [0088] each
R' is --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: [0089] two R'
on the same nitrogen are taken together with their intervening
atoms to form a 3-7 membered heterocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur; and [0090] each R is hydrogen, or an optionally substituted
group selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered
saturated or partially unsaturated carbocyclic ring, an 8-10
membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 3-7 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic
saturated or partially unsaturated heterocyclic ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
2. Definitions
[0091] Compounds of this disclosure include those described
generally above, and are further illustrated by the embodiments,
sub-embodiments, and species disclosed herein. As used herein, the
following definitions shall apply unless otherwise indicated. For
purposes of this disclosure, the chemical elements are identified
in accordance with the Periodic Table of the Elements, CAS version,
Handbook of Chemistry and Physics, 75.sup.th Ed. Additionally,
general principles of organic chemistry are described in "Organic
Chemistry," Thomas Sorrell, University Science Books, Sausalito:
1999, and "March's Advanced Organic Chemistry," 5.sup.th Ed., Ed.:
Smith, M. B. and March, J., John Wiley & Sons, New York: 2001,
the entire contents of which are hereby incorporated by reference
to the extent they are consistent herewith.
[0092] As described herein, compounds of the disclosure may
optionally be substituted with one or more substituents, such as
are illustrated generally above, or as exemplified by particular
classes, subclasses, and species of the disclosure. It will be
appreciated that the phrase "optionally substituted" is used
interchangeably with the phrase "substituted or unsubstituted." In
general, the term "substituted," whether preceded by the term
"optionally" or not, refers to the replacement of hydrogen radicals
in a given structure with the radical of a specified substituent.
Unless otherwise indicated, an optionally substituted group may
have a substituent at each substitutable position of the group, and
when more than one position in any given structure may be
substituted with more than one substituent selected from a
specified group, the substituent may be either the same or
different at every position. Combinations of substituents
envisioned by this disclosure are preferably those that result in
the formation of stable or chemically feasible compounds.
[0093] The term "stable," as used herein, refers to compounds that
are not substantially altered when subjected to conditions to allow
for their production, detection, and preferably their recovery,
purification, and use for one or more of the purposes disclosed
herein. In some embodiments, a stable compound or chemically
feasible compound is one that is not substantially altered when
kept at a temperature of 40.degree. C. or less, in the absence of
moisture or other chemically reactive conditions, for at least a
week.
[0094] The term "aliphatic" or "aliphatic group," as used herein,
means a straight-chain (i.e., unbranched) or branched, substituted
or unsubstituted hydrocarbon chain that is completely saturated or
that contains one or more units of unsaturation, or a monocyclic
hydrocarbon or bicyclic hydrocarbon that is completely saturated or
that contains one or more units of unsaturation, but which is not
aromatic (also referred to herein as "carbocycle" "cycloaliphatic"
or "cycloalkyl"), that has a single point of attachment to the rest
of the molecule. Unless otherwise specified, aliphatic groups
contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic
groups contain 1-6 aliphatic carbon atoms. In yet other embodiments
aliphatic groups contain 1-4 aliphatic carbon atoms. In some
embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl")
refers to a monocyclic C3-C8 hydrocarbon or bicyclic C.sub.8-C12
hydrocarbon that is completely saturated or that contains one or
more units of unsaturation, but which is not aromatic, that has a
single point of attachment to the rest of the molecule wherein any
individual ring in said bicyclic ring system has 3-7 members.
Suitable aliphatic groups include, but are not limited to, linear
or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl
groups and hybrids thereof such as (cycloalkyl)alkyl,
(cycloalkenyl)alkyl or (cycloalkyl)alkenyl. In other embodiments,
an aliphatic group may have two geminal hydrogen atoms replaced
with oxo (a bivalent carbonyl oxygen atom .dbd.O), or a
ring-forming substituent, such as --O-(straight or branched
alkylene or alkylene)-O-- to form an acetal or ketal.
[0095] In certain embodiments, exemplary aliphatic groups include,
but are not limited to, ethynyl, 2-propynyl, 1-propenyl, 2-butenyl,
1,3-butadienyl, 2-pentenyl, vinyl (ethenyl), allyl, isopropenyl,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, isopentyl, sec-pentyl, neo-pentyl, tert-pentyl,
cyclopentyl, hexyl, isohexyl, sec-hexyl, cyclohexyl,
2-methylpentyl, tert-hexyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl,
1,3-dimethylbutyl, and 2,3-dimethyl but-2-yl.
[0096] The term "heterocycle," "heterocyclyl,"
"heterocycloaliphatic," or "heterocyclic" as used herein means
non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in
which one or more ring members is an independently selected
heteroatom. In some embodiments, the "heterocycle," "heterocyclyl,"
"heterocycloaliphatic," or "heterocyclic" group has three to
fourteen ring members in which one or more ring members is a
heteroatom independently selected from oxygen, sulfur, nitrogen, or
phosphorus, and each ring in the system contains 3 to 7 ring
members.
[0097] A heterocyclic ring can be attached to its pendant group at
any heteroatom or carbon atom that results in a stable structure
and, when specified, any of the ring atoms can be optionally
substituted. Examples of such saturated or partially unsaturated
heterocyclic radicals include, without limitation,
tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl,
pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl,
dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and
quinuclidinyl.
[0098] The term "heteroatom" means one or more of oxygen, sulfur,
nitrogen, phosphorus, or silicon (including, any oxidized form of
nitrogen, sulfur, phosphorus, or silicon; the quaternized form of
any basic nitrogen or; a substitutable nitrogen of a heterocyclic
ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in
pyrrolidinyl) or NR.sup.+ (as in N-substituted pyrrolidinyl).
[0099] The term "unsaturated," as used herein, means that a moiety
has one or more units of unsaturation.
[0100] As used herein, the term "partially unsaturated" refers to a
ring moiety that includes at least one double or triple bond. The
term "partially unsaturated" is intended to encompass rings having
multiple sites of unsaturation, but is not intended to include aryl
or heteroaryl moieties, as herein defined.
[0101] The term "aryl" used alone or as part of a larger moiety as
in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic,
bicyclic, and tricyclic ring systems having a total of five to
fourteen ring members, wherein one or more ring in the system is
aromatic and wherein each ring in the system contains 3 to 7 ring
members. The term "aryl" may be used interchangeably with the term
"aryl ring". The term "aryl" also refers to heteroaryl ring systems
as defined herein below. In certain embodiments of the present
disclosure, "aryl" refers to an aromatic ring system which
includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl
and the like, which may bear one or more substituents. Also
included within the scope of the term "aryl," as it is used herein,
is a group in which an aromatic ring is fused to one or more
non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,
phenanthridinyl, or tetrahydronaphthyl, and the like.
[0102] The term "heteroaryl," used alone or as part of a larger
moiety as in "heteroaralkyl" or "heteroarylalkoxy," refers to
monocyclic, bicyclic, and tricyclic ring systems having a total of
five to fourteen ring members, wherein one or more ring in the
system is aromatic, one or more ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
Heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl,
pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,
oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,
pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,
naphthyridinyl, and pteridinyl.
[0103] The terms "heteroaryl" and "heteroar-," as used herein, also
include groups in which a heteroaromatic ring is fused to one or
more aryl, cycloaliphatic, or heterocyclyl rings. Exemplary
heteroaryl rings include indolyl, isoindolyl, benzothienyl,
benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,
benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,
quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,
phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, and
pyrido[2,3-b]-1,4-oxazin-3(4H)-one.
[0104] As described herein, compounds of the disclosure may contain
"optionally substituted" moieties. In general, the term
"substituted," whether preceded by the term "optionally" or not,
means that one or more hydrogens of the designated moiety are
replaced with a suitable substituent. Unless otherwise indicated,
an "optionally substituted" group may have a suitable substituent
at each substitutable position of the group, and when more than one
position in any given structure may be substituted with more than
one substituent selected from a specified group, the substituent
may be either the same or different at every position. Combinations
of substituents envisioned by this disclosure are preferably those
that result in the formation of stable or chemically feasible
compounds. The term "stable," as used herein, refers to compounds
that are not substantially altered when subjected to conditions to
allow for their production, detection, and, in certain embodiments,
their recovery, purification, and use for one or more of the
purposes disclosed herein.
[0105] Suitable monovalent substituents on a substitutable carbon
atom of an "optionally substituted" group are independently
halogen; --(CH.sub.2).sub.0-4R.sup..smallcircle.;
--(CH.sub.2).sub.0-4OR.sup..smallcircle.;
--O(CH2).sub.0-4R.sup..smallcircle.,
--O--(CH.sub.2).sub.0-4C(O)OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4CH(OR.sup..smallcircle.).sub.2;
--(CH.sub.2).sub.0-4SR.sup..smallcircle.; --(CH.sub.2).sub.0-4Ph,
which may be substituted with R.sup..smallcircle.;
--(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-4Ph which may be substituted
with R.sup..smallcircle.; --CH.dbd.CHPh, which may be substituted
with R.sup..smallcircle.;
--(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1-pyridyl which may be
substituted with R.sup..smallcircle.; --NO.sub.2; --CN; --N.sub.3;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.).sub.2;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)C(S)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)NR.sub.2;
--N(R.sup..smallcircle.)C(S)NR.sup..smallcircle..sub.2;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)OR.sup..smallcircle.;
--N(R.sup..smallcircle.)N(R.sup..smallcircle.)C(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)N(R.sup..smallcircle.)C(O)NR.sup..smallcircle..su-
b.2;
--N(R.sup..smallcircle.)N(R.sup..smallcircle.)C(O)OR.sup..smallcircle-
.; --(CH.sub.2).sub.0-4C(O)R.sup..smallcircle.;
--C(S)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)SR.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)OSiR.sub.3;
--(CH.sub.2).sub.0-4C(O)R.sup..smallcircle.;
--OC(O)(CH.sub.2).sub.0-4SR.sup..smallcircle.,
SC(S)SR.sup..smallcircle.;
--(CH.sub.2).sub.0-4SC(O)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)NR.sup..smallcircle..sub.2;
--C(S)NR.sup..smallcircle..sub.2; --C(S)SR.sup..smallcircle.;
--SC(S)SRO, --(CH.sub.2).sub.0-4OC(O)NR.sup..smallcircle..sub.2;
--C(O)N(OR.sup..smallcircle.)R.sup..smallcircle.;
--C(O)C(O)R.sup..smallcircle.;
--C(O)CH.sub.2C(O)R.sup..smallcircle.;
--C(NOR.sup..smallcircle.)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4SSR.sup..smallcircle.;
--(CH.sub.2).sub.0-4S(O).sub.2R.sup..smallcircle.;
--(CH.sub.2).sub.0- 4S(O).sub.2OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4S(O).sub.2R.sup..smallcircle.;
--S(O).sub.2NR.sup..smallcircle..sub.2;
--(CH.sub.2).sub.0-4S(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)S(O).sub.2NR.sup..smallcircle..sub.2;
--N(R.sup..smallcircle.)S(O).sub.2R.sup..smallcircle.;
--N(OR.sup..smallcircle.)R.sup..smallcircle.;
--C(NH)NR.sup..smallcircle..sub.2; --P(O).sub.2R.sup..smallcircle.;
--P(O)R.sup..smallcircle..sub.2; --OP(O)R.sup..smallcircle..sub.2;
--OP(O)(OR.sup..smallcircle.).sub.2; SiR.sup..smallcircle..sub.3;
--(C.sub.1-4 straight or branched
alkylene)O--N(R.sup..smallcircle.).sub.2; or --(C.sub.1-4 straight
or branched alkylene)C(O)O--N(R.sup..smallcircle.).sub.2, wherein
each R.sup..smallcircle. may be substituted as defined below and is
independently hydrogen, C.sub.1-6 aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, --CH.sub.2-(5-6 membered heteroaryl ring),
or a 5-6-membered saturated, partially unsaturated, or aryl ring
having 0-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or, notwithstanding the definition above, two
independent occurrences of R.sup..smallcircle., taken together with
their intervening atom(s), form a 3-12-membered saturated,
partially unsaturated, or aryl mono- or bicyclic ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, which may be substituted as defined below.
[0106] Suitable monovalent substituents on R.sup..smallcircle. (or
the ring formed by taking two independent occurrences of
R.sup..smallcircle. together with their intervening atoms), are
independently halogen, --(CH2)0-2R.sup..cndot.,
-(haloR.sup..cndot.), --(CH2)0-2OH, --(CH2)0-2OR.sup..cndot.,
--(CH2)0-2CH(OR.sup..cndot.)2; --O(haloR.sup..cndot.), --CN, --N3,
--(CH2)0-2C(O)R.sup..cndot., --(CH2)0-2C(O)OH,
--(CH2)0-2C(O)OR.sup..cndot., --(CH2)0-2SR.sup..cndot.,
--(CH2)0-2SH, --(CH2)0-2NH2, --(CH2)0-2NHR.sup..cndot.,
--(CH2)0-2NR.sup..cndot., --NO, --SiR.sup..cndot.,
--OSiR.sup..cndot., --C(O)SR.sup..cndot.--(C1-4 straight or
branched alkylene)C(O)OR.sup..cndot., or --SSR.sup..cndot. wherein
each R.sup..cndot. is unsubstituted or where preceded by "halo" is
substituted only with one or more halogens, and is independently
selected from C.sub.1-4 aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. Suitable divalent
substituents on a saturated carbon atom of R.sup..smallcircle.
include .dbd.O and .dbd.S.
[0107] Suitable divalent substituents on a saturated carbon atom of
an "optionally substituted" group include the following: .dbd.O,
.dbd.S, .dbd.NNR*.sub.2, .dbd.NNHC(O)R*, .dbd.NNHC(O)OR*,
.dbd.NNHS(O).sub.2R*, .dbd.NR*, .dbd.NOR*,
--O(C(R*.sub.2)).sub.2-3O--, or --S(C(R*.sub.2)).sub.2-3S--, and
.dbd.C(R*).sub.2, wherein each independent occurrence of R* is
selected from hydrogen, C.sub.1-6 aliphatic which may be
substituted as defined below, or an unsubstituted 5-6-membered
saturated, partially unsaturated, or aryl ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. Suitable divalent substituents that are bound to vicinal
substitutable carbons of an "optionally substituted" group include:
--O(CR*.sub.2).sub.2-3O--, wherein each independent occurrence of R
is selected from hydrogen, C.sub.1-6 aliphatic which may be
substituted as defined below, or an unsubstituted 5-6-membered
saturated, partially unsaturated, or aryl ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0108] Suitable substituents on the aliphatic group of R* include
halogen, --R.sup..cndot., -(haloR.sup..cndot.), --OH, --OR',
--O(haloR.sup..cndot.), --CN, --C(O)OH, --C(O)OR', --NH2,
--NHR.sup..cndot., --NR.sup..cndot., or --NO, wherein each
R.sup..cndot. is unsubstituted or where preceded by "halo" is
substituted only with one or more halogens, and is independently
C.sub.1-4 aliphatic, --CH.sub.2Ph, --O(CH.sub.2).sub.0-1Ph, or a
5-6-membered saturated, partially unsaturated, or aryl ring having
0-4 heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0109] Suitable substituents on a substitutable nitrogen of an
"optionally substituted" group include --R.sup..dagger.,
--NR.sup..dagger..sub.2, --C(O)R.sup..dagger.,
--C(O)OR.sup..dagger., --C(O)C(O)R.sup..dagger.,
--C(O)CH.sub.2C(O)R.sup..dagger., --S(O).sub.2R.sup..dagger.,
--S(O).sub.2NR.sup..dagger..sub.2, --C(S)NR.sup..dagger..sub.2,
--C(NH)NR.sup..dagger..sub.2, or
--N(R.sup..dagger.)S(O).sub.2R.sup..dagger.; wherein each
R.sup..dagger. is independently hydrogen, C.sub.1-6 aliphatic which
may be substituted as defined below, unsubstituted --OPh, or an
unsubstituted 5-6-membered saturated, partially unsaturated, or
aryl ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, or, notwithstanding the definition
above, two independent occurrences of R.sup..dagger., taken
together with their intervening atom(s) form an unsubstituted
3-12-membered saturated, partially unsaturated, or aryl mono- or
bicyclic ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0110] Suitable substituents on the aliphatic group of Rt are
independently halogen, --R.sup..cndot., -(haloR.sup..cndot.), --OH,
--OR.sup..cndot., --O(haloR.sup..cndot.), --CN, --C(O)OH,
--C(O)OR.sup..cndot., --NH.sup.2, --NHR.sup..cndot.,
--NR.sup..cndot., or --NO.sub.2, wherein each R.sup..cndot. is
unsubstituted or where preceded by "halo" is substituted only with
one or more halogens, and is independently C.sub.1-4 aliphatic,
--CH2Ph, --O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated,
partially unsaturated, or aryl ring having 0-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur.
[0111] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
disclosure.
[0112] Unless otherwise stated, all tautomeric forms of the
compounds of the disclosure are within the scope of the
disclosure.
[0113] Additionally, unless otherwise stated, structures depicted
herein are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms.
[0114] For example, compounds having the present structures except
for the replacement of hydrogen by deuterium or tritium, or the
replacement of a carbon by a .sup.11C- or .sup.13C- or
.sup.14C-enriched carbon are within the scope of this disclosure.
Such compounds are useful, for example, as analytical tools or
probes in biological assays.
3. Description of Exemplary Compounds
[0115] According to one embodiment, the present disclosure provides
a compound of formula I:
##STR00007##
or a pharmaceutically acceptable salt thereof, wherein: [0116] each
of Ring A and Ring B is independently an optionally substituted
group selected from phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; [0117] L.sup.1 is independently a covalent bond
or an optionally substituted bivalent C.sub.1-6 hydrocarbon chain,
wherein one or more methylene units of L.sup.1 are optionally and
independently replaced by --O--, --S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--OC(O)--, or --C(O)O--; [0118] R' is --R, --C(O)R, --CO.sub.2R, or
--SO.sub.2R, or: [0119] two R' on the same nitrogen are taken
together with their intervening atoms to form a 3-7 membered
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, and sulfur; and [0120] R is hydrogen, or an
optionally substituted group selected from C.sub.1-6 aliphatic,
phenyl, a 3-7 membered saturated or partially unsaturated
carbocyclic ring, an 8-10 membered bicyclic saturated, partially
unsaturated or aryl carbocyclic ring, a 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, an 8-10 membered
bicyclic saturated or partially unsaturated heterocyclic ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0121] According to one embodiment, the present disclosure provides
a compound of formula II:
##STR00008##
or a pharmaceutically acceptable salt thereof, wherein: [0122] each
of Ring A' and Ring B' is independently an optionally substituted
group selected from phenyl, an 8-10 membered bicyclic aryl ring, a
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, or an 8-10
membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; [0123]
L.sup.2 is --C(O)N(R')--; [0124] each R' is independently --R,
--C(O)R, --CO.sub.2R, or --SO.sub.2R, or: [0125] two R' on the same
nitrogen are taken together with their intervening atoms to form a
3-7 membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur; and [0126] each R is
hydrogen, or an optionally substituted group selected from C1-6
aliphatic, phenyl, a 3-7 membered saturated or partially
unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, an 8-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0127] According to one embodiment, the present disclosure provides
a compound of formula III:
##STR00009##
or a pharmaceutically acceptable salt thereof, wherein: [0128] Ring
C'' is independently an optionally substituted group selected from
phenyl, an 8-10 membered bicyclic aryl ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur; [0129] each of R.sup.1,
R.sup.2 and R.sup.3 is independently halogen, R', --C(O)R',
--C(S)R', --CO2R', --C(O)N(R')2, --C(S)N(R').sub.2, --S(O)R',
--SO.sub.2R', --SO.sub.2N(R').sub.2, --OR', --O--(C.sub.1-6
aliphatic)-N(R').sub.2, --O--(C.sub.1-6 aliphatic)-OR', --OC(O)R',
--SR', --NO.sub.2, --N(R').sub.2, --NR'C(O)R', --NR'C(O)OR',
--NR'C(O)N(R').sub.2, --NR'SO.sub.2R', --NR'SO.sub.2N(R').sub.2, or
--NR'OR'; [0130] a is 1-4; [0131] b is 1-5; [0132] X.sup.1 is
--C(R.sup.x).sub.2--, --NR.sup.x--, --NR.sup.xC(R.sup.x).sub.2-- or
--OC(R.sup.x).sub.2--; [0133] X.sup.2 is --C(R.sup.x).sub.2-- or
--NR.sup.x--; [0134] each R.sup.x is independently R', --(C.sub.1-6
aliphatic)-N(R').sub.2, or --(C.sub.1-6 aliphatic)-OR'; [0135] each
R' is independently --R, --C(O)R, --CO.sub.2R, or --SO2R, or:
[0136] two R' on the same nitrogen are taken together with their
intervening atoms to form a 3-7 membered heterocyclic ring having
1-2 heteroatoms independently selected from nitrogen, oxygen, and
sulfur; and [0137] each R is hydrogen, or an optionally substituted
group selected from C1-6 aliphatic, phenyl, a 3-7 membered
saturated or partially unsaturated carbocyclic ring, an 8-10
membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 3-7 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic
saturated or partially unsaturated heterocyclic ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0138] As generally defined above, Ring A is an optionally
substituted group selected from phenyl, a 3-7 membered saturated or
partially unsaturated carbocyclic ring, an 8-10 membered bicyclic
saturated, partially unsaturated or aryl carbocyclic ring, a 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur;
[0139] In some embodiments, Ring A is optionally substituted
phenyl. In some embodiments, Ring A is substituted phenyl. In some
embodiments, Ring A is phenyl. In some embodiments, Ring A is
phenyl substituted with one or more halogen atoms. In some
embodiments, Ring A is phenyl substituted with two halogen atoms.
In some embodiments, Ring A is 2-fluoro-5-chlorophenyl. In some
embodiments, Ring A is 2-fluoro-5-bromophenyl. In some embodiments,
Ring A is 3-chloro-4-flluorophenyl.
[0140] In some embodiments, Ring A is an optionally substituted 3-7
membered saturated or partially unsaturated carbocyclic ring. In
some embodiments, Ring A is an optionally substituted 3-7 membered
saturated carbocyclic ring. In some embodiments, Ring A is a
substituted 3-7 membered saturated carbocyclic ring. In some
embodiments, Ring A is an unsubstituted 3-7 membered saturated
carbocyclic ring. In some embodiments, Ring A is an optionally
substituted 3-7 membered unsaturated carbocyclic ring. In some
embodiments, Ring A is a substituted 3-7 membered unsaturated
carbocyclic ring. In some embodiments, Ring A is an unsubstituted
3-7 membered unsaturated carbocyclic ring.
[0141] In some embodiments, Ring A is an optionally substituted
cycloheptyl. In some embodiments, Ring A is an optionally
substituted cyclohexyl. In some embodiments, Ring A is an
optionally substituted cyclopentyl. In some embodiments, Ring A is
an optionally substituted cyclobutyl. In some embodiments, Ring A
is an optionally substituted cyclopropyl.
[0142] In some embodiments, Ring A is an optionally substituted
8-10 membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring. In some embodiments, Ring A is an optionally
substituted 8-10 membered bicyclic saturated carbocyclic ring. In
some embodiments, Ring A is an optionally substituted 8-10 membered
bicyclic partially unsaturated carbocyclic ring. In some
embodiments, Ring A is an optionally substituted 8-10 membered
bicyclic aryl ring.
[0143] In some embodiments, Ring A is an optionally substituted 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A is a substituted 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0144] In some embodiments, Ring A is an unsubstituted 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0145] In some embodiments, Ring A is an optionally substituted 5
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, Ring A is an optionally substituted 6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0146] In some embodiments, Ring A is an optionally substituted
5-membered monocyclic heteroaryl ring having 1 heteroatom selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring A is
selected from pyrrolyl, furanyl, or thienyl.
[0147] In some embodiments, Ring A is an optionally substituted
5-membered heteroaryl ring having 2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In certain embodiments,
Ring A is an optionally substituted 5-membered heteroaryl ring
having 1 nitrogen atom, and an additional heteroatom selected from
sulfur or oxygen. Exemplary Ring A groups include optionally
substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl,
oxazolyl or isoxazolyl.
[0148] In some embodiments, Ring A is a 6-membered heteroaryl ring
having 1-3 nitrogen atoms. In other embodiments, Ring A is an
optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring A is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring A is an optionally substituted 6-membered
heteroaryl ring having 1 nitrogen. Exemplary Ring A groups include
optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, or tetrazinyl.
[0149] In some embodiments, Ring A is an optionally substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring A is a substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring A is an unsubstituted
3-7 membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0150] In some embodiments, Ring A is an optionally substituted 6
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring A is an optionally
substituted 6 membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
or sulfur. In some embodiments, Ring A is an optionally substituted
6 membered partially unsaturated heterocyclic ring having 2 oxygen
atom. In some embodiments, Ring A is an optionally substituted 6
membered partially unsaturated heterocyclic ring having 2 oxygen
atom having the structure
##STR00010##
[0151] In certain embodiments, Ring A is a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In certain
embodiments, Ring A is oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl,
pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl,
oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl,
dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl,
thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl,
dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl,
oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl,
dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,
oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl,
dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl,
oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl,
imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl,
imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl,
dioxolanedionyl, oxathiolanedionyl, piperazinedionyl,
morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl,
tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl,
piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or
tetrahydrothiopyranyl. In some embodiments, Ring A is an optionally
substituted 5 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur. In some embodiments, two optional
substituents are taken together with their intervening atom(s) to
form a 3-12-membered saturated, partially unsaturated, or aryl
mono- or bicyclic ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, which may be
substituted. In some embodiments, Ring A is an optionally
substituted group selected from
##STR00011##
[0152] In certain embodiments, Ring A is an optionally substituted
5-6 membered partially unsaturated monocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring A is an optionally substituted
tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or
oxazolinyl group.
[0153] In some embodiments, Ring A is an optionally substituted
8-10 membered bicyclic saturated or partially unsaturated
heterocyclic ring having 1-4 heteroatoms independently selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring A is an
optionally substituted indolinyl. In some embodiments, Ring A is an
optionally substituted isoindolinyl. In some embodiments, Ring A is
an optionally substituted 1,2,3,4-tetrahydroquinoline. In some
embodiments, Ring A is an optionally substituted
1,2,3,4-tetrahydroisoquinoline.
[0154] In some embodiments, Ring A is an optionally substituted
group selected from or
##STR00012##
[0155] In certain embodiments, Ring A is an optionally substituted
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A is an optionally substituted 5,6-fused
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In other embodiments, Ring A is an
optionally substituted 5,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring A is an optionally substituted
5,6-fused heteroaryl ring having 1 heteroatom independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring A is an optionally substituted indolyl. In some embodiments,
Ring A is an optionally substituted azabicyclo[3.2.1]octanyl. In
certain embodiments, Ring A is an optionally substituted 5,6-fused
heteroaryl ring having 2 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In some embodiments, Ring A is an
optionally substituted azaindolyl. In some embodiments, Ring A is
an optionally substituted benzimidazolyl. In some embodiments, Ring
A is an optionally substituted benzothiazolyl. In some embodiments,
Ring A is an optionally substituted benzoxazolyl. In some
embodiments, Ring A is an optionally substituted indazolyl. In
certain embodiments, Ring A is an optionally substituted 5,6-fused
heteroaryl ring having 3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0156] In certain embodiments, Ring A is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring A is an optionally substituted 6,6-fused heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In other embodiments, Ring A is an optionally
substituted 6,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A is an optionally substituted quinolinyl. In
some embodiments, Ring A is an optionally substituted
isoquinolinyl. According to one aspect, Ring A is an optionally
substituted 6,6-fused heteroaryl ring having 2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A is a quinazoline or a quinoxaline.
[0157] In some embodiments, two substituents on Ring A are
optionally taken together with their intervening atoms to form an
optionally substituted, 3-7 membered saturated, partially
unsaturated or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring A are optionally taken together with their
intervening atoms to form an optionally substituted phenyl. In some
embodiments, two substituents on Ring A are optionally taken
together with their intervening atoms to form an optionally
substituted 3-7 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, two substituents on Ring A
are optionally taken together with their intervening atoms to form
an optionally substituted 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring A are optionally taken together with their
intervening atoms to form an optionally substituted 5-6 membered
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen or sulfur.
[0158] As generally defined above, Ring B is an optionally
substituted group selected from phenyl, a 3-7 membered saturated or
partially unsaturated carbocyclic ring, an 8-10 membered bicyclic
saturated, partially unsaturated or aryl carbocyclic ring, a 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur;
[0159] In some embodiments, Ring B is an optionally substituted
phenyl. In some embodiments, Ring B is a substituted phenyl. In
some embodiments, Ring B is phenyl. In some embodiments, Ring B is
a phenyl substituted with one or more halogen atoms. In some
embodiments, Ring B is a phenyl substituted with two halogen atoms.
In some embodiments, Ring B is 2-fluoro-5-chlorophenyl. In some
embodiments, Ring B is 2-fluoro-5-bromophenyl. In some embodiments,
Ring B is 3-chloro-4-flluorophenyl.
[0160] In some embodiments, Ring B is an optionally substituted 3-7
membered saturated or partially unsaturated carbocyclic ring. In
some embodiments, Ring B is an optionally substituted 3-7 membered
saturated carbocyclic ring. In some embodiments, Ring B is a
substituted 3-7 membered saturated carbocyclic ring. In some
embodiments, Ring B is an unsubstituted 3-7 membered saturated
carbocyclic ring. In some embodiments, Ring B is an optionally
substituted 3-7 membered unsaturated carbocyclic ring. In some
embodiments, Ring B is a substituted 3-7 membered unsaturated
carbocyclic ring. In some embodiments, Ring B is an unsubstituted
3-7 membered unsaturated carbocyclic ring.
[0161] In some embodiments, Ring B is an optionally substituted
cycloheptyl. In some embodiments, Ring B is an optionally
substituted cyclohexyl. In some embodiments, Ring B is an
optionally substituted cyclopentyl. In some embodiments, Ring B is
an optionally substituted cyclobutyl. In some embodiments, Ring B
is an optionally substituted cyclopropyl.
[0162] In some embodiments, Ring B is an optionally substituted
8-10 membered bicyclic saturated, partially unsaturated or aryl
carbocyclic ring. In some embodiments, Ring B is an optionally
substituted 8-10 membered bicyclic saturated carbocyclic ring. In
some embodiments, Ring B is an optionally substituted 8-10 membered
bicyclic partially unsaturated carbocyclic ring. In some
embodiments, Ring B is an optionally substituted 8-10 membered
bicyclic aryl ring.
[0163] In some embodiments, Ring B is an optionally substituted 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B is a substituted 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In some embodiments, Ring B is an
unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0164] In some embodiments, Ring B is an optionally substituted 5
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, Ring B is an optionally substituted 6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0165] In some embodiments, Ring B is an optionally substituted
5-membered monocyclic heteroaryl ring having 1 heteroatom selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring B is
selected from pyrrolyl, furanyl, or thienyl.
[0166] In some embodiments, Ring B is an optionally substituted
5-membered heteroaryl ring having 2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In certain embodiments,
Ring B is an optionally substituted 5-membered heteroaryl ring
having 1 nitrogen atom, and an additional heteroatom selected from
sulfur or oxygen. Exemplary Ring B groups include optionally
substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl,
oxazolyl or isoxazolyl.
[0167] In some embodiments, Ring B is a 6-membered heteroaryl ring
having 1-3 nitrogen atoms. In other embodiments, Ring B is an
optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring B is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring B is an optionally substituted 6-membered
heteroaryl ring having 1 nitrogen. Exemplary Ring B groups include
optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, or tetrazinyl.
[0168] In some embodiments, Ring B is an optionally substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring B is a substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring B is an unsubstituted
3-7 membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0169] In some embodiments, Ring B is an optionally substituted 6
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In some embodiments, Ring B is an optionally
substituted 6 membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
or sulfur. In some embodiments, Ring B is an optionally substituted
6 membered partially unsaturated heterocyclic ring having 2 oxygen
atom. In some embodiments, Ring B is an optionally substituted 6
membered partially unsaturated heterocyclic ring having 2 oxygen
atom having the structure
##STR00013##
[0170] In certain embodiments, Ring B is a 3-7 membered saturated
or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In certain
embodiments, Ring B is oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl,
pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl,
oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl,
dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl,
thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl,
dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl,
oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl,
dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,
oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl,
dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl,
oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl,
imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl,
imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl,
dioxolanedionyl, oxathiolanedionyl, piperazinedionyl,
morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl,
tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl,
piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or
tetrahydrothiopyranyl. In some embodiments, Ring B is an optionally
substituted 5 membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur.
[0171] In certain embodiments, Ring B is an optionally substituted
5-6 membered partially unsaturated monocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring B is an optionally substituted
tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or
oxazolinyl group.
[0172] In some embodiments, Ring B is an optionally substituted
8-10 membered bicyclic saturated or partially unsaturated
heterocyclic ring having 1-4 heteroatoms independently selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring B is an
optionally substituted indolinyl. In some embodiments, Ring B is an
optionally substituted isoindolinyl. In some embodiments, Ring B is
an optionally substituted 1,2,3,4-tetrahydroquinoline. In some
embodiments, Ring B is an optionally substituted
1,2,3,4-tetrahydroisoquinoline. In some embodiments, Ring B is an
optionally substituted group selected from
##STR00014##
[0173] In certain embodiments, Ring B is an optionally substituted
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B is an optionally substituted 5,6-fused
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In other embodiments, Ring B is an
optionally substituted 5,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring B is an optionally substituted
5,6-fused heteroaryl ring having 1 heteroatom independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring B is an optionally substituted indolyl. In some embodiments,
Ring B is an optionally substituted azabicyclo[3.2.1]octanyl. In
certain embodiments, Ring B is an optionally substituted 5,6-fused
heteroaryl ring having 2 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In some embodiments, Ring B is an
optionally substituted azaindolyl. In some embodiments, Ring B is
an optionally substituted benzimidazolyl. In some embodiments, Ring
B is an optionally substituted benzothiazolyl. In some embodiments,
Ring B is an optionally substituted benzoxazolyl. In some
embodiments, Ring B is an optionally substituted indazolyl. In
certain embodiments, Ring B is an optionally substituted 5,6-fused
heteroaryl ring having 3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0174] In certain embodiments, Ring B is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring B is an optionally substituted 6,6-fused heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In other embodiments, Ring B is an optionally
substituted 6,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B is an optionally substituted quinolinyl. In
some embodiments, Ring B is an optionally substituted
isoquinolinyl. According to one aspect, Ring B is an optionally
substituted 6,6-fused heteroaryl ring having 2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B is a quinazoline or a quinoxaline.
[0175] In some embodiments, two substituents on Ring B are
optionally taken together with their intervening atoms to form an
optionally substituted, 3-7 membered saturated, partially
unsaturated or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring B are optionally taken together with their
intervening atoms to form an optionally substituted phenyl. In some
embodiments, two substituents on Ring B are optionally taken
together with their intervening atoms to form an optionally
substituted 3-7 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, two substituents on Ring B
are optionally taken together with their intervening atoms to form
an optionally substituted 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring B are optionally taken together with their
intervening atoms to form an optionally substituted 5-6 membered
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen or sulfur.
[0176] As generally defined above, L.sup.1 is independently a
covalent bond or an optionally substituted bivalent C.sub.1-6
hydrocarbon chain, wherein one or more methylene units of L.sup.1
are optionally and independently replaced by --O--, --S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--OC(O)--, or --C(O)O--, wherein each R' is independently as
defined above and described herein.
[0177] In some embodiments, L.sup.1 is a covalent bond.
[0178] In some embodiments, L.sup.1 is an optionally substituted
bivalent C.sub.1-6 hydrocarbon chain, wherein one or more methylene
units of L.sup.1 are optionally and independently replaced by
--O--, --S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R')--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--N(R')S(O).sub.2--, --OC(O)--, or --C(O)O--, wherein each R' is
independently as defined above and described herein.
[0179] In some embodiments, L.sup.1 is --C(O)NR'--, wherein R' is
as defined above and described herein. In some embodiments, L.sup.1
is --C(O)NR--, wherein R is as defined above and described herein.
In some embodiments, L.sup.1 is --CONH--. In some embodiments,
L.sup.1 is --NHCO--.
[0180] In some embodiments, Ring A is an optionally substituted
group selected from
##STR00015##
Ring B is optionally substituted phenyl; and L.sup.1 is a covalent
bond.
[0181] As generally defined above, each R' is independently --R,
--C(O)R, --CO.sub.2R, or --SO.sub.2R, or: [0182] two R' on the same
nitrogen are taken together with their intervening atoms to form a
3-7 membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0183] In some embodiments, each R' is independently --R, --C(O)R,
--CO.sub.2R, or --SO.sub.2R, wherein R is as defined above and
described herein. In some embodiments, R' is R, wherein R is as
defined above and described herein. In some embodiments, R' is
hydrogen. In some embodiments, R' is
--(CH.sub.2).sub.1-6N(R).sub.2, wherein each R is independently as
defined above and described herein. In some embodiments, R' is
--(CH.sub.2).sub.2N(CH.sub.3).sub.2.
[0184] As generally defined above, each R is hydrogen, or an
optionally substituted group selected from C.sub.1-6 aliphatic,
phenyl, a 3-7 membered saturated or partially unsaturated
carbocyclic ring, an 8-10 membered bicyclic saturated, partially
unsaturated or aryl carbocyclic ring, a 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, an 8-10 membered
bicyclic saturated or partially unsaturated heterocyclic ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0185] In some embodiments, R is hydrogen.
[0186] In some embodiments, R is an optionally substituted group
selected from C.sub.1-6 aliphatic, phenyl, a 3-7 membered saturated
or partially unsaturated carbocyclic ring, an 8-10 membered
bicyclic saturated, partially unsaturated or aryl carbocyclic ring,
a 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments, R
is an optionally substituted C.sub.1-6 aliphatic. In some
embodiments, R is an optionally substituted phenyl. In some
embodiments, R is an optionally substituted 3-7 membered saturated
or partially unsaturated carbocyclic ring. In some embodiments, R
is an optionally substituted 8-10 membered bicyclic saturated,
partially unsaturated or aryl carbocyclic ring. In some
embodiments, R is an optionally substituted a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments, R
is an optionally substituted 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments, R
is an optionally substituted 8-10 membered bicyclic saturated or
partially unsaturated heterocyclic ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R is optionally substituted 8-10 membered bicyclic
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0187] In some embodiments, Ring A is an optionally substituted
group selected from phenyl, or an 8-10 membered bicyclic heteroaryl
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur; Ring B is an optionally substituted 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur; and L.sup.1 is --CONH--
or --NHCO--.
[0188] In some embodiments, Ring A is an optionally substituted
phenyl; Ring B is an optionally substituted 5 membered monocyclic
heteroaryl ring having two heteroatoms independently selected from
nitrogen, oxygen, or sulfur; and L.sup.1 is --CONH-- or
--NHCO--.
[0189] In some embodiments, Ring A is an optionally substituted
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; Ring B is
an optionally substituted 5 membered monocyclic heteroaryl ring
having two heteroatoms independently selected from nitrogen,
oxygen, or sulfur; and L.sup.1 is --CONH-- or --NHCO--.
[0190] According to one embodiment, the present disclosure provides
a compound of formula I having the structure of formula II:
##STR00016##
or a pharmaceutically acceptable salt thereof, wherein: [0191] each
of Ring A' and Ring B' is independently an optionally substituted
group selected from phenyl, an 8-10 membered bicyclic aryl ring, a
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, or an 8-10
membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; [0192]
L.sup.2 is --C(O)N(R')--; and wherein each of R' and R is
independently as defined above and described herein.
[0193] As generally defined above, Ring A' is independently an
optionally substituted group selected from phenyl, an 8-10 membered
bicyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0194] In some embodiments, Ring A' is an optionally substituted
phenyl. In some embodiments, Ring A' is a substituted phenyl. In
some embodiments, Ring A' is phenyl. In some embodiments, Ring A'
is a phenyl substituted with one or more halogen atoms. In some
embodiments, Ring A' is a phenyl substituted with two halogen
atoms. In some embodiments, Ring A' is 2-fluoro-5-chlorophenyl. In
some embodiments, Ring A' is 2-fluoro-5-bromophenyl.
[0195] In some embodiments, Ring A' is
3-chloro-4-flluorophenyl.
[0196] In some embodiments, Ring A' is an optionally substituted
8-10 membered bicyclic aryl ring. In some embodiments, Ring A' is
an optionally substituted 10 membered bicyclic aryl ring.
[0197] In some embodiments, Ring A' is an optionally substituted
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A' is a substituted 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0198] In some embodiments, Ring A' is an unsubstituted 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur.
[0199] In some embodiments, Ring A' is an optionally substituted 5
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, Ring A' is an optionally substituted 6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0200] In some embodiments, Ring A' is an optionally substituted
5-membered monocyclic heteroaryl ring having 1 heteroatom selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring A' is
selected from pyrrolyl, furanyl, or thienyl.
[0201] In some embodiments, Ring A' is an optionally substituted
5-membered heteroaryl ring having 2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In certain embodiments,
Ring A' is an optionally substituted 5-membered heteroaryl ring
having 1 nitrogen atom, and an additional heteroatom selected from
sulfur or oxygen. Exemplary Ring A' groups include optionally
substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl,
oxazolyl or isoxazolyl.
[0202] In some embodiments, Ring A' is a 6-membered heteroaryl ring
having 1-3 nitrogen atoms. In other embodiments, Ring A' is an
optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring A' is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring A' is an optionally substituted
6-membered heteroaryl ring having 1 nitrogen. Exemplary Ring A'
groups include optionally substituted pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
[0203] In certain embodiments, Ring A' is an optionally substituted
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A' is an optionally substituted 5,6-fused
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In other embodiments, Ring A' is an
optionally substituted 5,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring A' is an optionally
substituted 5,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A' is an optionally substituted indolyl. In some
embodiments, Ring A' is an optionally substituted
azabicyclo[3.2.1]octanyl. In certain embodiments, Ring A' is an
optionally substituted 5,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In some embodiments, Ring A' is an optionally substituted
azaindolyl. In some embodiments, Ring A' is an optionally
substituted benzimidazolyl. In some embodiments, Ring A' is an
optionally substituted benzothiazolyl. In some embodiments, Ring A'
is an optionally substituted benzoxazolyl. In some embodiments,
Ring A' is an optionally substituted indazolyl. In certain
embodiments, Ring A' is an optionally substituted 5,6-fused
heteroaryl ring having 3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0204] In certain embodiments, Ring A' is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring A' is an optionally substituted 6,6-fused heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In other embodiments, Ring A' is an optionally
substituted 6,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A' is an optionally substituted quinolinyl. In
some embodiments, Ring A' is an optionally substituted
isoquinolinyl. According to one aspect, Ring A' is an optionally
substituted 6,6-fused heteroaryl ring having 2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring A' is a quinazoline or a quinoxaline.
[0205] In some embodiments, two substituents on Ring A' are
optionally taken together with their intervening atoms to form an
optionally substituted, 3-7 membered saturated, partially
unsaturated or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring A' are optionally taken together with their
intervening atoms to form an optionally substituted phenyl. In some
embodiments, two substituents on Ring A' are optionally taken
together with their intervening atoms to form an optionally
substituted 3-7 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, two substituents on Ring A'
are optionally taken together with their intervening atoms to form
an optionally substituted 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring A' are optionally taken together with their
intervening atoms to form an optionally substituted 5-6 membered
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen or sulfur.
[0206] As generally defined above, Ring B' is independently an
optionally substituted group selected from phenyl, an 8-10 membered
bicyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
[0207] In some embodiments, Ring B' is an optionally substituted
phenyl. In some embodiments, Ring B' is a substituted phenyl. In
some embodiments, Ring B' is phenyl. In some embodiments, Ring B'
is a phenyl substituted with one or more halogen atoms. In some
embodiments, Ring B' is a phenyl substituted with two halogen
atoms. In some embodiments, Ring B' is 2-fluoro-5-chlorophenyl. In
some embodiments, Ring B' is 2-fluoro-5-bromophenyl. In some
embodiments, Ring B' is 3-chloro-4-flluorophenyl.
[0208] In some embodiments, Ring B' is an optionally substituted
8-10 membered bicyclic aryl ring. In some embodiments, Ring B' is
an optionally substituted 10 membered bicyclic aryl ring.
[0209] In some embodiments, Ring B' is an optionally substituted
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B' is a substituted 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In some embodiments, Ring B' is an
unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0210] In some embodiments, Ring B' is an optionally substituted 5
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, Ring B' is an optionally substituted 6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0211] In some embodiments, Ring B' is an optionally substituted
5-membered monocyclic heteroaryl ring having 1 heteroatom selected
from nitrogen, oxygen, or sulfur. In some embodiments, Ring B' is
selected from pyrrolyl, furanyl, or thienyl.
[0212] In some embodiments, Ring B' is an optionally substituted
5-membered heteroaryl ring having 2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In certain embodiments,
Ring B' is an optionally substituted 5-membered heteroaryl ring
having 1 nitrogen atom, and an additional heteroatom selected from
sulfur or oxygen. Exemplary Ring B' groups include optionally
substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl,
oxazolyl or isoxazolyl.
[0213] In some embodiments, Ring B' is a 6-membered heteroaryl ring
having 1-3 nitrogen atoms. In other embodiments, Ring B' is an
optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring B' is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring B' is an optionally substituted
6-membered heteroaryl ring having 1 nitrogen. Exemplary Ring B'
groups include optionally substituted pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
[0214] In certain embodiments, Ring B' is an optionally substituted
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B' is an optionally substituted 5,6-fused
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In other embodiments, Ring B' is an
optionally substituted 5,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In certain embodiments, Ring B' is an optionally
substituted 5,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B' is an optionally substituted indolyl. In some
embodiments, Ring B' is an optionally substituted
azabicyclo[3.2.1]octanyl. In certain embodiments, Ring B' is an
optionally substituted 5,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In some embodiments, Ring B' is an optionally substituted
azaindolyl. In some embodiments, Ring B' is an optionally
substituted benzimidazolyl. In some embodiments, Ring B' is an
optionally substituted benzothiazolyl. In some embodiments, Ring B'
is an optionally substituted benzoxazolyl. In some embodiments,
Ring B' is an optionally substituted indazolyl. In certain
embodiments, Ring B' is an optionally substituted 5,6-fused
heteroaryl ring having 3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0215] In certain embodiments, Ring B' is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring B' is an optionally substituted 6,6-fused heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In other embodiments, Ring B' is an optionally
substituted 6,6-fused heteroaryl ring having 1 heteroatom
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B' is an optionally substituted quinolinyl. In
some embodiments, Ring B' is an optionally substituted
isoquinolinyl. According to one aspect, Ring B' is an optionally
substituted 6,6-fused heteroaryl ring having 2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring B' is a quinazoline or a quinoxaline.
[0216] In some embodiments, two substituents on Ring B' are
optionally taken together with their intervening atoms to form an
optionally substituted, 3-7 membered saturated, partially
unsaturated or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring B' are optionally taken together with their
intervening atoms to form an optionally substituted phenyl. In some
embodiments, two substituents on Ring B' are optionally taken
together with their intervening atoms to form an optionally
substituted 3-7 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, two substituents on Ring B'
are optionally taken together with their intervening atoms to form
an optionally substituted 3-7 membered saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, two
substituents on Ring B' are optionally taken together with their
intervening atoms to form an optionally substituted 5-6 membered
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen or sulfur.
[0217] As generally defined above, L.sup.2 is --C(O)N(R')--,
wherein R' is as defined above and described herein. In some
embodiments, L.sup.2 is --C(O)NH--. In some embodiments, L.sup.2 is
--C(O)N(R)--, wherein R is as defined above and described herein.
In some embodiments, Ring A' is directly connected to the carbonyl
group in L.sup.2. In some embodiments, Ring B' is directly
connected to the carbonyl group in L.sup.2.
[0218] In some embodiments, the present disclosure provides a
compound of formula II having the structure of formula II-a:
##STR00017##
or a pharmaceutically acceptable salt thereof, wherein: [0219] Ring
A'' is an optionally substituted phenyl or benzimidazolyl ring;
[0220] Ring B'' is an optionally substituted 5-6 membered
heteroaryl ring having 1-2 heteroatoms independently selected from
nitrogen, oxygen, or sulfur; [0221] L.sup.3 is --C(O)NH--; [0222]
R.sup.4 is halogen or R; [0223] R.sup.5 is an optionally
substituted C.sub.1-6 aliphatic; [0224] each of d and e is
independently 0-5; and [0225] wherein R is as defined above and
described herein.
[0226] As generally defined above, Ring A'' is an optionally
substituted phenyl or benzimidazolyl ring.
[0227] In some embodiments, Ring A'' is optionally substituted
phenyl. In some embodiments, Ring A'' is unsubstituted phenyl. In
some embodiments, Ring A'' is substituted phenyl. In some
embodiments, Ring A'' is 3-chloro-4-fluorophenyl. In some
embodiments, Ring A'' is optionally substituted benzimidazolyl. In
some embodiments, Ring A'' is unsubstituted benzimidazolyl. In some
embodiments, Ring A'' is substituted benzimidazolyl.
[0228] As generally defined above, Ring B'' is an optionally
substituted 5-6 membered heteroaryl ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur.
[0229] In some embodiments, Ring B'' is an optionally substituted 5
membered heteroaryl ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen or sulfur. In some embodiments, Ring
B'' is unsubstituted 5 membered heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen or sulfur.
In some embodiments, Ring B'' is substituted 5 membered heteroaryl
ring having 1-2 heteroatoms independently selected from nitrogen,
oxygen or sulfur. In certain embodiments, Ring B'' is an optionally
substituted 5-membered heteroaryl ring having 1 nitrogen atom, and
an additional heteroatom selected from sulfur or oxygen. Exemplary
Ring B'' groups include optionally substituted pyrazolyl,
imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.
[0230] In some embodiments, Ring B'' is a 6-membered heteroaryl
ring having 1-3 nitrogen atoms. In other embodiments, Ring B'' is
an optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring B'' is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring B'' is an optionally substituted
6-membered heteroaryl ring having 1 nitrogen. Exemplary Ring B''
groups include optionally substituted pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
[0231] As generally defined above, each R.sup.4 is independently
halogen or R, wherein R is as defined above and described herein.
In some embodiments, R.sup.4 is halogen. In some embodiments,
R.sup.4 is --F. In some embodiments, R.sup.4 is --Cl. In some
embodiments, R.sup.4 is --Br. In some embodiments, R.sup.4 is --I.
In some embodiments, R.sup.4 is R, wherein R is as defined above
and described herein.
[0232] As generally defined above, each R.sup.5 is independently an
optionally substituted C.sub.1-6 aliphatic. In some embodiments,
R.sup.5 is optionally substituted straight or branched C.sub.1-6
alkyl. In some embodiments, R.sup.5 is optionally substituted
hexyl, pentyl, butyl, propyl, ethyl or methyl. In some embodiments,
R.sup.5 is hexyl. In some embodiments, R.sup.5 is pentyl. In some
embodiments, R.sup.5 is butyl. In some embodiments, R.sup.5 is
propyl. In some embodiments, R.sup.5 is ethyl. In some embodiments,
R.sup.5 is methyl. In some embodiments, R.sup.5 is isopropyl.
[0233] In some embodiments, R.sup.5 is optionally substituted
C.sub.1-6 cycloalkyl. In some embodiments, R.sup.5 is optionally
substituted cyclohexyl. In some embodiments, R.sup.5 is optionally
substituted cyclopentyl. In some embodiments, R.sup.5 is optionally
substituted cyclobutyl. In some embodiments, R.sup.5 is optionally
substituted cyclopropyl. In some embodiments, R.sup.5 is
1-hydroxycyclobutyl. In some embodiments, R.sup.5 is cyclohexyl,
cyclopentyl, cyclobutyl, or cyclopropyl.
[0234] As generally defined above, each of d and e is independently
0-5. In some embodiments, d is 0. In some embodiments, d is 1. In
some embodiments, d is 2. In some embodiments, d is 3. In some
embodiments, d is 4. In some embodiments, d is 5. In some
embodiments, e is 0. In some embodiments, e is 1. In some
embodiments, e is 2. In some embodiments, e is 3. In some
embodiments, e is 4. In some embodiments, e is 5.
[0235] In some embodiments, a compound of formula II-a is selected
from
##STR00018##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0236] According to one embodiment, the present disclosure provides
a compound of formula I having the structure of formula III:
##STR00019##
or a pharmaceutically acceptable salt thereof, wherein: [0237] Ring
C'' is independently an optionally substituted group selected from
phenyl, an 8-10 membered bicyclic aryl ring, a 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur; [0238] each of R.sup.1,
R.sup.2 and R.sup.3 is independently halogen, R', --C(O)R',
--C(S)R', --CO.sub.2R', --C(O)N(R').sub.2, --C(S)N(R').sub.2,
--S(O)R', --SO.sub.2R', --SO.sub.2N(R').sub.2, --OR',
--O--(C.sub.1-6 aliphatic)-N(R').sub.2, --O--(C.sub.1-6
aliphatic)-OR', --OC(O)R', --SR', --NO.sub.2, --N(R').sub.2,
--NR'C(O)R', --NR'C(O)OR', --NR'C(O)N(R').sub.2, --NR'SO.sub.2R',
--NR'SO.sub.2N(R').sub.2, or --NR'OR'; [0239] a is 1-4; [0240] b is
1-5; [0241] X.sup.1 is --C(R.sup.x).sub.2--, --NR.sup.x--,
--NR.sup.xC(R.sup.x).sub.2-- or --OC(R.sup.x).sub.2--; [0242]
X.sup.2 is --C(R.sup.x).sub.2-- or --NR.sup.x--; [0243] each
R.sup.x is independently R', --(C.sub.1-6 aliphatic)-N(R').sub.2,
or --(C.sub.1-6 aliphatic)-OR'; [0244] each R' is independently
--R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: [0245] two R' on the
same nitrogen are taken together with their intervening atoms to
form a 3-7 membered heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, and sulfur; and
[0246] each R is hydrogen, or an optionally substituted group
selected from C1-6 aliphatic, phenyl, a 3-7 membered saturated or
partially unsaturated carbocyclic ring, an 8-10 membered bicyclic
saturated, partially unsaturated or aryl carbocyclic ring, a 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur, a 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, an 8-10 membered bicyclic saturated or partially
unsaturated heterocyclic ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8-10 membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0247] In some embodiments, Ring C'' is optionally substituted
phenyl. In some embodiments, Ring C'' is substituted phenyl. In
some embodiments, Ring C'' is phenyl. In some embodiments, Ring C''
is phenyl substituted with one or more halogen atoms. In some
embodiments, Ring C'' is phenyl substituted with two halogen atoms.
In some embodiments, Ring C'' is 2-fluoro-5-chlorophenyl. In some
embodiments, Ring C'' is 2-fluoro-5-bromophenyl. In some
embodiments, Ring C'' is 3-chloro-4-flluorophenyl.
[0248] In some embodiments, Ring C'' is an optionally substituted
8-10 membered bicyclic aryl ring.
[0249] In some embodiments, Ring C'' is an optionally substituted
5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring C'' is an optionally substituted 5 membered
heteroaryl ring having 1-2 heteroatoms independently selected from
nitrogen, oxygen or sulfur. In some embodiments, Ring C'' is
unsubstituted 5 membered heteroaryl ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, Ring C'' is substituted 5 membered heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen, oxygen
or sulfur. In certain embodiments, Ring C'' is an optionally
substituted 5-membered heteroaryl ring having 1 nitrogen atom, and
an additional heteroatom selected from sulfur or oxygen. Exemplary
Ring C'' groups include optionally substituted pyrazolyl,
imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.
[0250] In some embodiments, Ring C'' is a 6-membered heteroaryl
ring having 1-3 nitrogen atoms. In other embodiments, Ring C'' is
an optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, Ring C'' is an optionally
substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In
certain embodiments, Ring C'' is an optionally substituted
6-membered heteroaryl ring having 1 nitrogen. Exemplary Ring C''
groups include optionally substituted pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl. In some
embodiments, Ring C'' is optionally substituted pyridinyl.
[0251] In certain embodiments, Ring C'' is an optionally
substituted 8-10 membered bicyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In some embodiments, Ring C'' is an optionally substituted
5,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. In other embodiments,
Ring C'' is an optionally substituted 5,6-fused heteroaryl ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur. In certain embodiments, Ring C'' is an
optionally substituted 5,6-fused heteroaryl ring having 1
heteroatom independently selected from nitrogen, oxygen, or sulfur.
In some embodiments, Ring C'' is an optionally substituted indolyl.
In some embodiments, Ring C'' is an optionally substituted
benzofuranyl. In some embodiments, Ring C'' is an optionally
substituted azabicyclo[3.2.1]octanyl. In certain embodiments, Ring
C'' is an optionally substituted 5,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In some embodiments, Ring C'' is an optionally substituted
azaindolyl. In some embodiments, Ring C'' is an optionally
substituted benzimidazolyl. In some embodiments, Ring C'' is an
optionally substituted benzothiazolyl. In some embodiments, Ring
C'' is an optionally substituted benzoxazolyl. In some embodiments,
Ring C'' is an optionally substituted indazolyl. In certain
embodiments, Ring C'' is an optionally substituted 5,6-fused
heteroaryl ring having 3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0252] In certain embodiments, Ring C'' is an optionally
substituted 6,6-fused heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, Ring C'' is an optionally substituted 6,6-fused
heteroaryl ring having 1-2 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. In other embodiments, Ring C'' is an
optionally substituted 6,6-fused heteroaryl ring having 1
heteroatom independently selected from nitrogen, oxygen, or sulfur.
In some embodiments, Ring C'' is an optionally substituted
quinolinyl. In some embodiments, Ring C'' is an optionally
substituted isoquinolinyl. According to one aspect, Ring C'' is an
optionally substituted 6,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, or
sulfur. In some embodiments, Ring C'' is a quinazoline or a
quinoxaline.
[0253] As generally defined above, each R.sup.1 is independently
halogen, R', --C(O)R', --C(S)R', --CO2R', --C(O)N(R')2,
--C(S)N(R')2, --S(O)R', --SO2R', --SO2N(R')2, --OR', --O--(C1-6
aliphatic)-N(R').sub.2, --O--(C.sub.1-6 aliphatic)-OR', --OC(O)R',
--SR', --NO.sub.2, --N(R').sub.2, --NR'C(O)R', --NR'C(O)OR',
--NR'C(O)N(R').sub.2, --NR'SO.sub.2R', --NR'SO.sub.2N(R').sub.2, or
--NR'OR', wherein each R' is independently as defined above and
described herein.
[0254] In some embodiments, R.sup.1 is --OR', wherein R' is as
defined above and described herein. In some embodiments, R.sup.1 is
OR, wherein R is as defined above and described herein. In some
embodiments, R.sup.1 is --OMe.
[0255] In some embodiments, R.sup.1 is --O--(C.sub.1-6
aliphatic)-OR', wherein R' is as defined above and described
herein. In some embodiments, R.sup.1 is --O--(C.sub.1-6
aliphatic)-OR, wherein R is as defined above and described herein.
In some embodiments, R.sup.1 is --O--(C1-6 aliphatic)-OH. In some
embodiments, R.sup.1 is --O(CH2)2OH. In some embodiments, R.sup.1
is --O--(C.sub.1-6 aliphatic)-OR, wherein R is C.sub.1-6 alkyl. In
some embodiments, R.sup.1 is --O(CH.sub.2).sub.2OMe.
[0256] In some embodiments, R.sup.1 is --O--(C.sub.1-6
aliphatic)-N(R').sub.2, wherein each R' is independently as defined
above and described herein. In some embodiments, R.sup.1 is
--O--(C.sub.1-6 aliphatic)-N(R').sub.2, wherein two R' on the same
nitrogen are taken together with their intervening atoms to form a
3-7 membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments,
R.sup.1 is --O--(C.sub.1-6 aliphatic)-N(R).sub.2, wherein each R is
independently C.sub.1-6 alkyl. In some embodiments, R.sup.1 is
--O(CH.sub.2).sub.2N(R').sub.2, wherein each R' is independently
C.sub.1-6 alkyl. In some embodiments, R.sup.1 is
--O(CH.sub.2).sub.2N(R').sub.2, wherein two R' on the same nitrogen
are taken together with their intervening atoms to form a 3-7
membered heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments,
R.sup.1 is
##STR00020##
[0257] As generally defined above, each R.sup.2 is independently
halogen, R', --C(O)R', --C(S)R', --CO.sub.2R', --C(O)N(R').sub.2,
--C(S)N(R').sub.2, --S(O)R', --SO.sub.2R', --SO.sub.2N(R').sub.2,
--OR', --O--(C.sub.1-6 aliphatic)-N(R')2, --O--(C1-6
aliphatic)-OR', --OC(O)R', --SR', --NO2, --N(R')2, --NR'C(O)R',
--NR'C(O)OR', --NR'C(O)N(R').sub.2, --NR'SO.sub.2R',
--NR'SO.sub.2N(R').sub.2, or --NR'OR', wherein each R' is
independently as defined above and described herein.
[0258] In some embodiments, R.sup.2 is halogen. In some
embodiments, R.sup.2 is --F. In some embodiments, R.sup.2 is --Cl.
In some embodiments, R.sup.2 is --Br. In some embodiments, R.sup.2
is --I.
[0259] As generally defined above, each R.sup.3 is independently
halogen, R', --C(O)R', --C(S)R', --CO.sub.2R', --C(O)N(R').sub.2,
--C(S)N(R').sub.2, --S(O)R', --SO.sub.2R', --SO.sub.2N(R').sub.2,
--OR', --O--(C.sub.1-6 aliphatic)-N(R').sub.2, --O--(C.sub.1-6
aliphatic)-OR', --OC(O)R', --SR', --NO.sub.2, --N(R').sub.2,
--NR'C(O)R', --NR'C(O)OR', --NR'C(O)N(R').sub.2, --NR'SO.sub.2R',
--NR'SO.sub.2N(R').sub.2, or --NR'OR', wherein each R' is
independently as defined above and described herein.
[0260] In some embodiments, R.sup.3 is hydrogen.
[0261] As generally defined above, a is 1-4. In some embodiments, a
is 1. In some embodiments, a is 2. In some embodiments, a is 4. In
some embodiments, a is 4.
[0262] In some embodiments, a=3 and each R.sup.1 is --OMe.
[0263] As generally defined above, b is 1-5. In some embodiments, b
is 1. In some embodiments, b is 2. In some embodiments, b is 3. In
some embodiments, b is 4. In some embodiments, b is 5.
[0264] As generally defined above, X.sup.1 is --C(R.sup.x).sub.2--,
--NR.sup.x--, --NR.sup.xC(R.sup.x).sub.2-- or
--OC(R.sup.x).sub.2--, wherein each R.sup.X is independently as
defined above and described herein. In some embodiments, X.sup.1 is
--C(R.sup.x).sub.2--, wherein each R.sup.x is independently as
defined above and described herein. In some embodiments, X.sup.1 is
--CH.sub.2--. In some embodiments, X.sup.1 is --NR.sup.x--, wherein
R' is as defined above and described herein. In some embodiments,
X.sup.1 is --NH--. In some embodiments, X.sup.1 is
--NR.sup.xC(R.sup.x).sub.2--, wherein R' is independently as
defined above and described herein. In some embodiments, X.sup.1 is
--NHCH.sub.2--. In some embodiments, R' is --OC(R.sup.x).sub.2--,
wherein R' is independently as defined above and described herein.
In some embodiments, R' is --OCH.sub.2--.
[0265] In some embodiments, X.sup.1 is --NR'--, wherein R' is as
defined above and described herein. In some embodiments, X.sup.1 is
--NR'--, wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, X.sup.1 is --NR'--, wherein R' is unsubstituted
C.sub.1-6 alkyl. In some embodiments, X.sup.1 is --NR'-- wherein R'
is methyl. In some embodiments, X.sup.1 is --NR'-- wherein R' is
propyl. In some embodiments, X.sup.1 is --NR'-- wherein R' is
n-propyl. In some embodiments, X.sup.1 is --NR'--, wherein R' is
methyl. In some embodiments, X.sup.1 is --NR'--, wherein R' is
substituted C.sub.1-6 alkyl.
[0266] In some embodiments, X.sup.1 is --NR.sup.x--, wherein
R.sup.x is wherein R.sup.x is --(C.sub.1-6 aliphatic)-N(R').sub.2,
wherein each R' is independently as defined above and described
herein. In some embodiments, --NR.sup.x-- is
--(CH.sub.2).sub.1-6N(R').sub.2, wherein each R' is independently
as defined above and described herein. In some embodiments,
--NR.sup.x-- is --(CH.sub.2).sub.1-6N(R').sub.2, wherein each R' is
optionally substituted C.sub.1-6 alkyl. In some embodiments,
--NR.sup.x-- is --(CH.sub.2).sub.2N(R').sub.2, wherein each R' is
optionally substituted C.sub.1-6 alkyl. In some embodiments,
X.sup.1 is --N[(CH.sub.2).sub.2N(CH.sub.3).sub.2]--.
[0267] As generally defined above, X.sup.2 is --C(R.sup.x).sub.2--
or --NR.sup.x--, wherein each R.sup.x is independently as defined
above and described herein. In some embodiments, X.sup.2 is
--C(R').sub.2--, wherein each R' is independently as defined above
and described herein. In some embodiments, X.sup.2 is --CH.sub.2--.
In some embodiments, X.sup.2 is --NR'--, wherein R' is as defined
above and described herein. In some embodiments, X.sup.2 is
--NH--.
[0268] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-a:
##STR00021##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described herein. In
some embodiments, the present disclosure provides a compound of
formula III having the structure of formula III-b:
##STR00022##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0269] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-c:
##STR00023##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0270] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-d:
##STR00024##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0271] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-e:
##STR00025##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0272] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-f:
##STR00026##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0273] In some embodiments, the present disclosure provides a
compound of formula III having the structure of formula III-g:
##STR00027##
or a pharmaceutically acceptable salt thereof, wherein each
variable is independently as defined above and described
herein.
[0274] Exemplary compounds are set forth in Table 1, below.
TABLE-US-00001 TABLE 1 Exemplary Compounds ##STR00028## 1
##STR00029## 2 ##STR00030## 3 ##STR00031## 4 ##STR00032## 5
##STR00033## 6 ##STR00034## 7 ##STR00035## 8 ##STR00036## 9
##STR00037## 10 ##STR00038## 11 ##STR00039## 12 ##STR00040## 13
##STR00041## 14 ##STR00042## 15 ##STR00043## 16 ##STR00044## 17
##STR00045## 18 ##STR00046## 19 ##STR00047## 20 ##STR00048## 21
##STR00049## 22 ##STR00050## 23 ##STR00051## 24 ##STR00052## 25
##STR00053## 26 ##STR00054## 27 ##STR00055## 28 ##STR00056## 29
##STR00057## 30 ##STR00058## 31 ##STR00059## 32 ##STR00060## 33
##STR00061## 34 ##STR00062## 35 ##STR00063## 36 ##STR00064## 37
##STR00065## 38 ##STR00066## 39 ##STR00067## 40 ##STR00068## 41
##STR00069## 42 ##STR00070## 43 ##STR00071## 44 ##STR00072## 45
##STR00073## 46 ##STR00074## 47 ##STR00075## 48 ##STR00076## 49
##STR00077## 50 ##STR00078## 51 ##STR00079## 52 ##STR00080## 53
##STR00081## 54 ##STR00082## 55 ##STR00083## 56 ##STR00084## 57
##STR00085## 58 ##STR00086## 59 ##STR00087## 60 ##STR00088## 61
##STR00089## 62 ##STR00090## 63 ##STR00091## 64 ##STR00092## 65
##STR00093## 66 ##STR00094## 67 ##STR00095## 68 ##STR00096## 69
##STR00097## 70 ##STR00098## 71 ##STR00099## 72 ##STR00100## 73
##STR00101## 74 ##STR00102## 75 ##STR00103## 76 ##STR00104## 77
##STR00105## 78 ##STR00106## 79 ##STR00107## 80 ##STR00108## 81
##STR00109## 82 ##STR00110## 83 ##STR00111## 84 ##STR00112## 85
##STR00113## 86 ##STR00114## 87 ##STR00115## 88 ##STR00116## 89
##STR00117## 90 ##STR00118## 91 ##STR00119## 92 ##STR00120## 93
##STR00121## 94 ##STR00122## 95 ##STR00123## 96 ##STR00124## 97
##STR00125## 98 ##STR00126## 99 ##STR00127## 100 ##STR00128## 101
##STR00129## 102 ##STR00130## 103 ##STR00131## 104 ##STR00132## 105
##STR00133## 106 ##STR00134## 107 ##STR00135## 108 ##STR00136## 109
##STR00137## 110 ##STR00138## 111 ##STR00139## 112 ##STR00140## 113
##STR00141## 114 ##STR00142## 115 ##STR00143## 116 ##STR00144## 117
##STR00145## 118 ##STR00146## 119 ##STR00147## 120 ##STR00148## 121
##STR00149## 122 ##STR00150## 123
##STR00151## 124 ##STR00152## 125 ##STR00153## 126 ##STR00154## 127
##STR00155## 128 ##STR00156## 129 ##STR00157## 130 ##STR00158## 131
##STR00159## 132 ##STR00160## 133 ##STR00161## 134 ##STR00162## 135
##STR00163## 136
[0275] In some embodiments, the present disclosure provides a
compound depicted in Table 1, above, or a pharmaceutically
acceptable salt thereof.
4. General Methods of Providing the Present Compounds
[0276] The compounds of this disclosure may be prepared or isolated
in general by synthetic and/or semi-synthetic methods known to
those skilled in the art for analogous compounds.
Pharmaceutically Acceptable Compositions
[0277] It will be appreciated that certain of the compounds of
present disclosure can exist in free form for treatment, or where
appropriate, as a pharmaceutically acceptable salt thereof.
[0278] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. A "pharmaceutically acceptable salt" means any
non-toxic salt or salt of an ester of a compound of this disclosure
that, upon administration to a recipient, is capable of providing,
either directly or indirectly, a compound of this disclosure or a
pharmaceutically active metabolite or residue thereof. As used
herein, the term "pharmaceutically active metabolite or residue
thereof" means that a metabolite or residue thereof is also a
pharmaceutically active compound in accordance with the present
disclosure.
[0279] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al., describes pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66,
1-19, incorporated herein by reference to the extent it is
consistent herewith. Pharmaceutically acceptable salts of the
compounds of this disclosure include those derived from suitable
inorganic and organic acids and bases. Examples of pharmaceutically
acceptable, nontoxic acid addition salts are salts of an amino
group formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid and perchloric
acid or with organic acids such as acetic acid, oxalic acid, maleic
acid, tartaric acid, citric acid, succinic acid or malonic acid or
by using other methods used in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C1-4 alkyl)4 salts. This
disclosure also envisions the quaternization of any basic
nitrogen-containing groups of the compounds disclosed herein. Water
or oil-soluble or dispersable products may be obtained by such
quaternization. Representative alkali or alkaline earth metal salts
include sodium, lithium, potassium, calcium, magnesium, and the
like. Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and
aryl sulfonate.
[0280] In some cases, compounds of the present disclosure may
contain one or more acidic functional groups and, thus, may be
capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. The term
"pharmaceutically-acceptable salts" in these instances refers to
the relatively non-toxic, inorganic and organic base addition salts
of compounds of the present disclosure. These salts can likewise be
prepared in situ in the administration vehicle or the dosage form
manufacturing process, or by separately reacting the purified
compound in its free acid form with a suitable base, such as the
hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a
pharmaceutically-acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethyldiamine, ethanolamine, diethanolamine, piperazine and the
like.
[0281] According to another aspect of the present disclosure,
pharmaceutically acceptable compositions are provided, wherein
these compositions comprise any of the compounds as described
herein, and optionally comprise a pharmaceutically acceptable
carrier, adjuvant or vehicle, which, as used herein, includes any
and all solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
[0282] Remington's Pharmaceutical Sciences, Sixteenth Edition, E.
W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses
various carriers used in formulating pharmaceutically acceptable
compositions and known techniques for the preparation thereof.
Except insofar as any conventional carrier medium is incompatible
with the compounds of the disclosure, such as by producing any
undesirable biological effect or otherwise interacting in a
deleterious manner with any other component(s) of the
pharmaceutically acceptable composition, its use is contemplated to
be within the scope of this disclosure. Some examples of materials
which can serve as pharmaceutically acceptable carriers include,
but are not limited to, ion exchangers, alumina, aluminum stearate,
lecithin, serum proteins, such as human serum albumin, buffer
substances such as phosphates, glycine, sorbic acid, or potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water, salts or electrolytes, such as protamine sulfate,
disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl pyrrolidone, polyacrylates, waxes,
polyethyl-polyoxypropyl-block polymers, wool fat, sugars such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propyl glycol or polyethyl glycol; esters such
as ethyl oleate and ethyl laurate; agar; buffering agents such as
magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator. In some embodiments, the
compositions of the present disclosure additionally comprise one or
more of DMSO, PEG400, Tween-80, and hydropropyl beta cyclodextrin.
In some embodiments, the compositions of the present disclosure
additionally comprise 2% DMSO, 2% PEG400, 0.2% Tween80, and 20%
hydropropyl beta cyclodextrin.
[0283] The compositions provided by the present disclosure can be
employed in combination therapies, meaning that the present
compositions can be administered concurrently with, prior to, or
subsequent to, one or more other desired therapeutic agents or
medical procedures. The particular combination of therapies
(therapeutic agents or procedures) to employ in a combination
regimen will take into account compatibility of the desired
therapeutic agents and/or procedures and the desired therapeutic
effect to be achieved. It will also be appreciated that the
therapies employed may achieve a desired effect for the same
disorder (for example, a compound described herein may be
administered concurrently with another therapeutic agent used to
treat the same disorder), or they may achieve different effects
(e.g., control of any adverse effects).
[0284] The amount of additional therapeutic agent present in the
compositions of this disclosure will be no more than the amount
that would normally be administered in a composition comprising
that therapeutic agent as the only active agent. In certain
embodiments, the amount of additional therapeutic agent in the
present compositions will range from about 50% to 100% of the
amount normally present in a composition comprising that agent as
the only therapeutically active agent.
[0285] In an alternate embodiment, the methods of this disclosure
that utilize compositions that do not contain an additional
therapeutic agent, comprise the additional step of separately
administering to said patient an additional therapeutic agent. When
these additional therapeutic agents are administered separately
they may be administered to the patient prior to, sequentially with
or following administration of the compositions of this
disclosure.
[0286] The pharmaceutically acceptable compositions of this
disclosure can be administered to humans and other animals orally,
rectally, parenterally, intravenously, intracisternally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, or drops), bucally, as an oral or nasal spray, or the
like, depending on the severity of the disorder being treated.
[0287] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyl
glycol, 1,3-butyl glycol, dimethylformamide, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),
glycerol, tetrahydrofurfuryl alcohol, polyethyl glycols and fatty
acid esters of sorbitan, and mixtures thereof. Besides inert
diluents, the oral compositions can also include adjuvants such as
wetting agents, emulsifying and suspending agents, sweetening,
flavoring, and perfuming agents.
[0288] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0289] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0290] In order to prolong the effect of a compound of the present
disclosure, it is often desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by
dissolving or suspending the compound in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the
compound in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of compound to
polymer and the nature of the particular polymer employed, the rate
of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0291] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this disclosure with suitable non-irritating
excipients or carriers such as cocoa butter, polyethyl glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0292] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with one or more inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethyl glycols, sodium lauryl sulfate,
and mixtures thereof. In the case of capsules, tablets and pills,
the dosage form may also comprise buffering agents.
[0293] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethyl glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polyethyl
glycols and the like.
[0294] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms, the
active compound may be admixed with one or more inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0295] Dosage forms for topical or transdermal administration of a
compound of this disclosure include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, and
eye drops are also contemplated as being within the scope of this
disclosure. Additionally, the present disclosure contemplates the
use of transdermal patches, which have the added advantage of
providing controlled delivery of a compound to the body. Such
dosage forms can be made by dissolving or dispensing the compound
in the proper medium. Absorption enhancers can also be used to
increase the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel.
Uses of Compounds and Pharmaceutically Acceptable Compositions
[0296] The compounds of the disclosure are preferably formulated in
dosage unit form for ease of administration and uniformity of
dosage. The expression "dosage unit form" as used herein refers to
a physically discrete unit of agent appropriate for the patient to
be treated. It will be understood, however, that the total daily
usage of the compounds and compositions of the present disclosure
(also referred to herein as "therapeutically effective amount")
will be decided by the attending physician within the scope of
sound medical judgment. More particularly, as used herein, the
phrase "therapeutically effective amount" of the compound used in
the methods of the present disclosure refers to a sufficient amount
of a compound to treat SMA as defined herein, at a reasonable
benefit/risk ratio applicable to any medical treatment. It can be
understood, however, that the total daily usage of the compound and
pharmaceutically acceptable compositions including the compound for
use in the methods of the present disclosure can be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
patient can depend upon a variety of factors including the loss of
motor neuron function episode being treated and the severity of the
episode; activity of the specific compound employed; the specific
pharmaceutically acceptable composition employed; the age, body
weight, general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of
the compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well-known in the medical arts. For example, it is
well within the skill of the art to start doses of the compound at
levels lower than required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved.
[0297] In certain embodiments, the compounds of the disclosure may
be administered orally or parenterally at dosage levels of about
0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to
about 25 mg/kg, of subject body weight per day, one or more times a
day, to obtain the desired therapeutic effect.
[0298] The term "patient," as used herein, means an animal,
preferably a mammal, and most preferably a human. Particularly, the
patient refers to a subject that is susceptible to or has SMA. As
used herein, "susceptible to" refers to having little resistance to
a certain disease, disorder or condition, and in particular, to
SMA, including being genetically predisposed, having a family
history of, and/or having symptoms of the disease, disorder or
condition. Accordingly, in some embodiments, the compounds and/or
pharmaceutically acceptable compositions can be administered to a
subset of subjects in need of preventing/minimizing/controlling
loss of motor neuron function, progressive motor weakness, muscle
wasting, and paralysis. Some subjects that are in specific need of
restored/maintained motor neuron function may include patients who
are susceptible to, or at elevated risk of, experiencing loss of
motor neuron function, including subjects susceptible to, or at
elevated risk of, areflexia, muscle weakness, poor muscle tone,
muscle wasting, paralysis, fasciculations of the tongue, difficulty
sucking or swallowing, arthrogryposis, low weight, and the like. In
one particular embodiment, the methods can be administered to a
patient who has, or is susceptible to, or at elevated risk of, SMA.
Subjects may be susceptible to, or at elevated risk of,
experiencing SMA, and generally, loss of motor neuron function,
areflexia, muscle weakness, poor muscle tone, muscle wasting,
paralysis, fasciculations of the tongue, difficulty sucking or
swallowing, arthrogryposis, low weight due to family history, age,
environment, and/or lifestyle. Based on the foregoing, because some
of the method embodiments of the present disclosure are directed to
specific subsets or subclasses of identified subjects (that is, the
subset or subclass of patients susceptible to one or more specific
conditions noted herein), not all subjects will fall within the
subset or subclass of subjects as described herein for certain
diseases, disorders or conditions.
[0299] Various functions and advantages of these and other
embodiments of the present disclosure will be more fully understood
from the examples described below. The following examples are
intended to illustrate the benefits of the present disclosure, but
do not exemplify the full scope of the disclosure.
EXAMPLES
Example 1
[0300] In this Example, the assay method of the present disclosure
was utilized to identify compounds that increase SMN2
expression.
Materials and Methods
Cloning
[0301] The luciferase minigene from previous reporter vectors
SMN1-luc (T-luc) or SMN2-luc (C-luc) was shortened by digestion
with Sma I and Swa I to remove 2 kB from intron 6. The SMN exon 1-5
fragment was generated by PCR from human cDNA (exon 1 forward:
ccacaaatgtgggagggcgataacc (SEQ ID NO: 1) and exon 6 reverse:
tatctcgagtggtccagaaggaaatggaggcagcc (SEQ ID NO: 2)). The SMN
promoter elements were from p3.4.sup.T and p3.4.sup.C SMN (Monani
et al., Promoter analysis of the human centromeric and telomeric
survival motor neuron genes (SMNC and SMNT), Biochim Biophys Acta
1999, 1445 (3), 330-336). These were combined into pIRES cloning
vector (BD Clontech) at the multiple cloning site. The entire
reporter fragment was excised from pIRES and ligated into a pCEP4
(Invitrogen) plasmid that also expressed renilla luciferase from
nucleotides 299-1259 of phRL-null (Promega) from the CMV
promoter.
Cell Culture
[0302] Cells were incubated at 37.degree. C. with 5% CO2. HEK-293
cells were cultured in D-MEM (GIBCO 11995) with 10% fetal bovine
serum (FBS, ATLAS) and 1.times. pen-strep (GIBCO 15140). Reporter
cell lines containing SMN1, SMN2, or control luciferase reporter
were selected and maintained in D-MEM with 10% FBS and 1.times.
pen-strep with 200 .mu.g/mL hygromycin B (Invitrogen 10687-010).
3813 and 3814 fibroblasts were cultured in D-MEM (GIBCO 11995) with
15% fetal bovine serum (FBS, ATLAS) and 1.times. pen-strep (GIBCO
15140).
Luciferase Reporter Assay
[0303] The reporter cell lines were plated at 50,000 cells per well
in 96-well plates and incubated overnight. Compounds were added to
each well and incubated at 37.degree. C. overnight. The final DMSO
concentration was 0.1%. Luciferase activity was assayed using
either STEADYGLO (PROMEGA E2510) or DUALGLO (PROMEGA E2920)
luciferase using the WALLAC ENVISION multilabel reader. For
detailed assay conditions see Table 2A. All data points were
transformed from CPS to percent increase over basal expression in
the treated control wells (DMSO or H2O as appropriate).
Protein Detection
[0304] For analysis of SMN-luciferase fusion, cells were treated
with compound or DMSO for 25 hours. Cells were lysed with protein
lysis buffer (100 mM Tris pH 8.0, 100 mM NaCl, 0.1% NP-40, 8.0 M
urea, and protease inhibitor). Each sample was separated on a 10%
SDS-page gel, transferred to Immobilon-P membrane (MILLIPORE
IVPH00010) and blotted for the SMN-luciferase fusion with
anti-luciferase antibody (PROMEGA, #G7541), SMN with the 4f11 mouse
monoclonal antibody (described in Mattis et al., "Detection of
human survival motor neuron (SMN) protein in mice containing the
SMN2 transgene: Applicability to preclinical therapy development
for spinal muscular atrophy" J. Neurosci Methods, 2008), HA-tag
with 12CA5 monoclonal, actin (Sigma A2066) or a-tubulin (DM1a;
Sigma T6199).
[0305] For detection of SMN protein in patient fibroblasts, 8,000
cells per cm were plated 24 hours prior to drug addition. Fresh
media and compound were added every 24 hours. After 72 hours, cells
were harvested, washed with cold PBS, and lysed as above. It has
been determined that 10 .mu.g total protein per lane is within the
linear range for immunoblot detection of SMN and a-tubulin. Western
blots were probed for SMN with the 4f11 mouse monoclonal antibody
and a-tubulin.
[0306] Quantification of protein was performed with FUJIFILM
LAS-4000 Multifunctional Imaging System. The signal intensity was
measured for each band on an immunoblot, normalized to the loading
control, and the fold increase was determined in relation to the
appropriate DMSO treated control.
Overexpression Assays
[0307] Cells were plated at a density of 2.times.10.sup.6 per 60 cm
dish and incubated overnight. Cells were transfected with up to 6 g
of HA-tagged expression vector using FUGENE 6 at a 3:1 fugene:DNA
ratio and incubated overnight. After 24 hours cells were re-plated
at 1.times.10.sup.5 cells per well in a 96-well plate or
1.times.10.sup.6 cells per well in 6-well dishes. Cells were tested
24 to 48 hours later. Luciferase was assayed using DUALGLO
luciferase as described above. Protein lysates and RNA samples were
collected using protein lysis buffer or Trizol respectively.
Protein was analyzed by western blot and RNA was analyzed by
qRT-PCR.
PCR and RT-PCR
[0308] Compounds were tested at three concentrations that display
maximal activity in the luciferase assay. Cells were treated as
described above for the luciferase assay. Cells were harvested by
trypsinization, neutralized with trypsin inhibitor, and washed. RNA
was isolated from the cells using TRIZOL Reagent (Invitrogen
15596-026). cDNA was generated using the IMPROM-II Reverse
Transcription System (Promega A3801).
[0309] The forward primer pair recognizes the exon 5-6 junction,
which includes a restriction site that was engineered into the
reporter and will exclude amplification of endogenous SMN mRNA. The
reverse primers recognize either exon 7 or luciferase for detection
of full-length or total SMN-luciferase transcripts respectively.
For a reference control, cDNA from the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified. The
primers used include SMN exon5-forward (catttccttctggaccactcgag)
(SEQ ID NO: 3), luciferase-reverse (atagcttctgccaaccgaacgg) (SEQ ID
NO: 4), exon7-reverse (taaggaatgtgagcaccttccttc) (SEQ ID NO: 5),
GAPDH-reverse (G3A) (tccaccaccctgttgctgta) (SEQ ID NO: 6) and
GAPDH-forward (G3S) (accacagtccatgccatcac) (SEQ ID NO: 7). qPCR was
performed as described in the protocol for iQ SYBRGREEN SUPERMIX
(Biorad 170-8882) using an EPPENDORF Mastercycler ep realplex.sup.4
Thermo Cycler. Reactions were incubated for a 10 minute, 94.degree.
C. hot start followed by 45 cycles of the following: 94.degree. C.
for 45 second, 60.degree. C. for 15 seconds, 72.degree. C. for 45
seconds. Melting curves for each reaction were obtained. Each
sample was assayed in triplicate and every plate contained a
5-point cDNA dilution course to calculate amplification efficiency
for each primer pair. The Pfaffl method was used to determine the
change in transcript levels relative to the DMSO and normalized to
GAPDH (Pfaffl, M. W., "A new mathematical model for relative
quantification in real-time RT-PCR," Nucleic Acids Res 2001, 29(9),
3e45).
Screening Protocol
[0310] Cells were plated in phenol red-free D-MEM (Gibco 21063)
with sodium pyruvate (Gibco 11360) and 10% FBS in the absence of
hygromycin B and pen-strep and allowed to adhere 1 hour prior to
addition of compound. Each compound was added to a single well with
a BECKMAN COULTER BIOMEK FX 384 to a final concentration of
approximately 2 M from compound stocks, based on an average
molecular weight of 500. The final DMSO concentration in test and
control wells was 0.13%. Plates were sealed with porous paper tape
and incubated for 24 hours. Luciferase expression was measured
using the STEADYLITE luciferase (Perkin Elmer 6016981) substrate
and the LJL Analyst HT, and read for counts per second (CPS) at an
integration time of 100,000 .mu.S. For comparison, data points were
transformed from CPS to percent activation over basal expression in
the DMSO treated control wells. This is summarized in Table 2B.
Library Composition
[0311] A compound library of approximately 115,000 small molecules,
including compounds approved by the Food and Drug Administration
(FDA), a purified natural products library, compounds purchased
from PEAKDALE (High Peak, UK), MAYBRIDGE Plc. (Cornwall, UK), Cerep
(Paris, France), BIONET RESEARCH Ltd. (Cornwall, UK), PRESTWICK
(Ilkirch, France), SPECS and BIOSPECS (CP Rijswijk, the
Netherlands), ENAMINE (Kiev, Ukraine), I.F. Lab LTD (Burlington,
Canada), and Chemical Diversity Labs (San Diego, Calif.), and small
molecules from academic institutions was tested. Compounds were
selected from vendors by applying a series of filters including
clogP and predicted solubility. All of the small molecules
generally adhere to Lipinski's rules (Lipinski et al. "Experimental
and computational approaches to estimate solubility and
permeability in drug discover and development settings," Adv Drug
Deliv Rev 2001, 46(1-3), 3-26) and contain a low proportion of
known toxicophores (i.e. Michael acceptors and alkylating agents)
and unwanted functionalities (i.e. imines, thiols, and quarternary
amines) and have been optimized for molecular diversity. Compound
source plates for the assay were prepared by spotting 0.4 .mu.l of
1.67 mM compound in DMSO in each well of a Greiner 384-well plate,
with columns 23 and 24 spotted with neat DMSO for positive and
negative controls. These were then sealed with aluminum plate seals
and stored at -20.degree. C.
Results
Development of a New Reporter
[0312] Over time, the original C33a reporter cell lines displayed a
decrease in the difference in luciferase signal from SMN1 and SMN2
(FIG. 7) and showed inconsistent responses to treatment with drugs
known to increase SMN expression (data not shown). These original
reporters were also driven by the CMV promoter and displayed much
higher basal levels of SMN1- and SMN2-luciferase activity in the
absence of treatment (FIG. 7), which could diminish the reporters'
ability to detect compounds that are less potent and more
selective. To address this, a new reporter assay was designed that
would be more responsive to molecular cues that regulate the levels
of SMN expression through multiple pathways. The SMN promoter and
exon 7 splicing cassette were combined into a single construct to
simultaneously identify compounds that increase SMN transcription
or exon 7 inclusion (FIGS. 1A-1D). The presence of the native SMN
promoter may also influence recruitment of splicing factors to the
transcript and better reproduce the context in which the endogenous
gene is processed and expressed. Since a subset of compounds might
stabilize the SMN RNA or protein, for example, by interfering with
its metabolism or ubiquitination, exons 1-5 were included in the
new reporter. This reporter produces a full-length SMN-luciferase
fusion protein that should be regulated and metabolized in a manner
that is more consistent with the endogenous SMN protein. The entire
reporter was cloned into an Epstein Barr Virus (EBV) vector that is
maintained autonomously as stable episomes in some human cell
lines.
[0313] To summarize, the new assay design: i) replaced the CMV
promoter with 3.4 kb of the SMN1 or SMN2 promoters with an intact
transcription start site; ii) included the cDNA for exons 1-5
following the authentic promoter and translational ATG; iii)
deleted a portion of intron 6 since the size of the entire 6 kb
intron complicated cloning; iv) included the exon 7 splicing
cassette with the firefly luciferase reporter; v) cloned renilla
luciferase expressed from the CMV promoter into the construct for
monitoring copy number, cell viability and specificity of the
transcriptional effects; and vi) transferred the 9 kb reporter SMN1
and SMN2 cassettes into EBV ori based pCEP4. The result is
reporters with sequences from SMN1 and SMN2 in the context of their
respective promoters. These constructs, each .about.19 kb, will
produce increased SMN-luciferase fusion protein if there is an
induction of 1) transcription from the SMN promoter; 2) inclusion
of exon 7; or 3) stabilization of the SMN-luciferase mRNA or fusion
protein. Not only will this reporter be able to identify potential
positive modifiers of SMN expression, but the design of the screen
and the renilla control also reduces the likelihood of selecting
compounds that are toxic or cause non-specific increases in
transcription. These reporters were transfected into HEK 293 cells.
These human cells are easy to culture and expand, highly
transfectable, and maintain EBV based plasmids extrachromosomally.
Stable clonal cell lines were isolated that express the full-length
SMN-luciferase protein as appropriate in both the SMN1-luc or
SMN2-luc reporters.
Analysis of SMN1-Luc and SMN2-Luc Clonal Cell Lines Population
[0314] Luciferase expression was analyzed in the stable HEK293 cell
lines containing the SMN1- and SMN2-luciferase reporters. In the
initial stable mixed cell populations, mixed SMN1-luc cells
displayed 30% more luciferase activity than the mixed SMN2-luc
cells. A pair of clonal cell lines was isolated, clonal SMN1-luc
and clonal-SMN2-luc, in which the clonal-SMN1-luc cell line has
50-fold higher luciferase activity when compared to clonal-SMN2-luc
cell line (FIG. 2A). This range of expression provides a large
window for potential activation of SMN2 expression with drug
treatment. Since these reporters were designed for use in either
high or low throughput screens, the dynamic range of activation is
very important. The selection of SMN2-luc clones that maintain low
levels of basal SMN-luciferase expression was instrumental in
establishing this range of activation and was a dramatic
improvement over not only the original C33a based reporter cell
lines but also the mix population HEK293 SMN1-luc and SMN2-luc cell
lines (FIG. 7).
[0315] Expression of the SMN-luciferase protein fusion was
confirmed by western blot (FIG. 2B). All cells express endogenous
SMN (38 kD) but only the SMN1-luc cell line expresses detectable
levels of the SMN-luciferase fusion protein. The SMN-luciferase
fusion protein could only be detected in the SMN2-luc cell line
upon induction with compounds that increase SMN expression (SAHA
and sodium butyrate in FIG. 3B).
[0316] These clonal cell lines display the expected patterns of
exon 7 inclusion in the reporter transcripts for both the SMN1-luc
and SMN2-luc cell lines. Published studies have determined that 90%
of transcripts from SMN1 include exon 7, while only 10-20% of
transcripts from SMN2 include exon 7. In FIG. 2C, end-point RT-PCR
for each cell line is shown and inclusion of exon 7 was calculated
as 64% for SMN1-luc and 13% for SMN2-luc. By quantitative reverse
transcription-PCR (qRT-PCR) using the primers illustrated in FIG.
4A, the percent inclusion for transcripts in each reporter was
calculated more precisely; 95.1%.+-.6.7 for SMN1-luc and
10.2%.+-.0.9 for SMN2-luc. Using these primers, the number of
copies of each reporter in these clonal cell lines was estimated.
SMN1-luc has 1 copy of the episomal reporter per cell and the
SMN2-luc cell line has about 10 copies of the reporter per
cell.
[0317] To determine the half-life of the SMN-fusion protein in
these cell lines, cells were treated with cycloheximide and assayed
for residual luciferase activity for 24 hours. The luciferase
activity in the SMN2-luc cell line had a t.sub.1/2 of 3.2 hours
(FIG. 2D). This matches well with published data for endogenous SMN
protein. This data suggests that any changes in protein stability
for the SMN-luciferase fusion protein would be easily detected
within 24 to 48 hours.
[0318] When the cell-lines were tested for tolerance to DMSO, it
was found the luciferase expression was virtually unchanged when
the final DMSO concentration in the reaction ranged from 0-1%.
Concentrations above 1% decreased luciferase activity and were very
likely detrimental to cell viability. Compounds are routinely
screened at a final DMSO concentration equal to or less than 0.1%.
Basal activity and response to compounds did not vary with serial
cell passage and was very reproducible after the clones were thawed
from liquid nitrogen storage.
SMN-Luciferase Reporter Cell Response to Compound Treatment.
[0319] In this assay, cells were treated with compounds previously
shown to increase SMN2 protein levels and luciferase expression
read for counts per second (CPS) at an integration time of 100,000
.mu.S (see Table 2A for detailed assay conditions). For comparison,
all data points are expressed as percent increase over basal
luciferase expression in the control cells.
[0320] Compounds that were previously shown to increase SMN
expression including SAHA, sodium butyrate, aclarubicin, TSA,
indoprofen, and tobramycin were tested in the cell lines (FIG. 3A).
These compounds were screened in both the SMN1-luc and SMN2-luc
cell lines and % increase of luciferase activity was plotted for
SMN1-luciferase, SMN2-luciferase, and the CMV driven internal
renilla luciferase control. The activity for each compound, except
tobramycin, matched or exceeded the published activities for these
compounds (FIG. 3A and Table 4). Tobramycin displayed no response
in these cell lines. Tobramycin is an aminoglycoside that increases
stable SMN protein levels from the SMN2 gene through
transcriptional read-through of the termination codon in exon 8. By
design, the reporter requires the inclusion of exon 7 and the
frameshift mutation therein to restore the reading frame for
luciferase. In the absence of exon 7, luciferase is out of frame.
Read-through will not correct the frame shift, so tobramycin cannot
and did not increase SMN-luciferase protein levels in the
SMN-luciferase reporter cell lines.
[0321] Changes in the SMN-luciferase fusion protein levels were
confirmed with SAHA in both the SMN1-luc and SMN2-luc cell lines
and with sodium butyrate in the SMN2-luc cell line. There was a
moderate increase in SMN-luciferase protein in the SMN1-luc cell
line with increasing SAHA (FIG. 3B), which corresponds to the
increase in luciferase activity (FIG. 3C). A greater than 9-fold
increase in SMN-luciferase protein was observed when the SMN2-luc
cell line was treated with either SAHA or sodium butyrate (FIG.
3B). This protein increase corresponds to an increase in luciferase
activity (FIG. 3C). These data confirm the correlation between
luciferase activity and levels of full-length SMN-luciferase
protein.
Analysis of mRNA in SMN2-Luciferase Reporter by qRT-PCR
[0322] Based on the new reporter design, it was predicted that this
screen could detect increased SMN-luciferase protein levels caused
by compounds that stimulate SMN2 transcription, exon 7 inclusion,
or by increasing the half-life of the SMN mRNA or protein.
Quantitative reverse transcriptase PCR (qRT-PCR) was used to
analyze SMN-luciferase mRNAs. All assays used RNA from control and
compound-treated SMN2-luc cells and primers pairs were chosen to
amplify only the SMN-luciferase derived transcripts (FIG. 4A). A
pair of primers were used that specifically detects both
full-length (exon 7 included) and .DELTA.7 (exon 7 excluded)
SMN-luciferase transcripts (primers 1 and 3). To measure changes in
exon 7 splicing efficiency, a primer pair was used that detects
only the full-length (exon 7 included) SMN-luciferase transcript
(primers 1 and 2).
[0323] For each sample, the percent increase in the amount of total
SMN-luciferase transcripts (gray bar) and exon 7 included
full-length SMN-luciferase transcripts (white bar) was plotted
(FIG. 4). Compounds that increase transcription should show
increased total SMN-luciferase transcripts (gray bar) with a
proportional increase for exon 7 included transcripts (white bar),
assuming that proper splicing of exon 7 is not rate limiting.
Compounds that stimulate exon 7 inclusion should increase the
amount of exon 7 included full-length transcript (white bar)
detected with little to no change in the expression of total
SMN-luciferase transcripts (gray bar). In these experiments, cells
were treated with compound, harvested, and each cell pellet was
divided for isolation of RNA and to assay luciferase activity. Each
qRT-PCR sample was normalized to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Pfaffl method
was used to calculate the change in the amount of total SMN mRNA
and determine whether compound treatments increased SMN
transcripts.
[0324] With the pan HDAC inhibitor compounds SAHA, TSA, valproic
acid, and sodium butyrate, increases in both the total and exon 7
included full-length SMN-luciferase transcripts were observed. The
increase in total SMN-luciferase mRNA suggests that these compounds
are increasing transcription, as would be expected for HDAC
inhibitors. These compounds also display a dramatic increase in
exon 7 included full-length transcripts that was greater in
magnitude than the increase of total SMN-luciferase transcripts.
This suggests that the HDAC inhibitors stimulate both SMN
transcription and exon 7 recognition and inclusion. With
aclarubicin at 300 nM, there was a potent increase in total
SMN-luciferase transcript. This increase was accompanied by a
lesser increase in exon 7 included transcript, indicating that
aclarubicin increases transcription of SMN2. In a cell in which
transcription is increased and splicing efficiency is unchanged,
the percent increase in exon 7 included transcripts (gray bar)
would be equal to the increase in total transcripts (white bar). In
this case, the amount of exon 7 included transcripts has decreased
even as the number of total transcripts increased. While
aclarubicin does increase transcription, it appears to antagonize
exon 7 recognition and inclusion. No consistent change in
transcript levels was observed with indoprofen.
Genetic Modulation of SMN-Luciferase Protein Expression.
[0325] To confirm that the reporter could also respond to
protein-induced changes in SMN expression, the splicing factors
SF2/ASF and Tra2P.beta. were overexpressed. SF2/ASF recognizes a
splicing enhancer that includes C in the +6 position at the 5' end
of exon 7 in SMN1 to promote exon 7 inclusion. This interaction is
antagonized by hnRNPA1, which binds at an overlapping splicing
silencer that is created by the C to T transition at the +6
position in exon 7 in the SMN2 transcripts. Little to no increase
in luciferase activity or change in RNA was observed in the
SMN2-luciferase reporter cell-line (FIGS. 5A and C). This was
expected, since hnRNPA1 has a high affinity for the silencing
element in exon 7 of SMN2. Even at its highest levels of
overexpression, SF2/ASF was unable to effectively stimulate the
weakened enhancer element (FIG. 5B).
[0326] Tra2.beta. is known to increase exon 7 inclusion in SMN2.
Despite low levels of Tra2.beta. expression (FIG. 5B), transfected
Tra2 stimulated luciferase activity 2-3 fold greater than the
negative control (FIG. 5A). With increasing amounts of Tra2.beta.
expressed there was up to a 6-fold increase in exon 7 included
transcripts (white bar) with only a 1.4-fold increase in total
transcripts (gray bar) (FIG. 5C). Analysis of the reporter mRNA
confirmed that this SMN2-luciferase reporter detects increased
inclusion of exon 7. From these data, the efficiency of exon 7
splicing of the reporter construct was calculated. Inclusion of
exon 7 was 6.5% in the negative control sample, and no increase was
observed with heterologous SF2/ASF expression. Exon 7 inclusion
increased to 20% with the highest amount of transfected Tra2P. This
validates the use of the SMN2-luciferase reporter cells to detect
and quantify changes in exon 7 inclusion as well as changes in SMN
expression in response to drug treatment and protein
overexpression.
Assay Validation for High-Throughput
[0327] To evaluate the suitability of the SMN2-luc cells for HTS,
test plates in both 96-well and 384-well format were prepared to
determine signal strength, well-to-well percent coefficient of
variation (% CV), amplitude of drug response, optimal cell density,
and time of treatment. It was observed that these HEK derived cell
lines displayed signal variability at low cell densities. As can be
seen in Table 3, the correlation between luciferase signal and cell
number was not linear. This is most clear in the 384-well format.
At low cell densities, these cells grow slowly and are less
responsive to treatment. A cell density of 50,000 or 10,000 per
well was found to be optimal in the 96- and 384-well plates,
respectively. To assess responsiveness, 500 g/mL sodium butyrate
was selected as a stimulus. While sodium butyrate is an HDAC
inhibitor that can cause non-specific transcriptional activation,
it has been reported to stimulate SMN2 transcription and SMN2 exon
7 inclusion. Cells were treated for 24 or 48 hours and while
increases in overall signal intensity were observed at the 48 hour
time point, there was no difference in the % activation with sodium
butyrate over control. However, there was an increase in the
well-to-well % CV. Therefore 24 hours was chosen as the optimal
time point for treatment. In the 96-well format, luciferase
activity in the SMN2-luc cell increased by more than 3 fold with
sodium butyrate and well-to-well % CV was 5% for both treated and
control cells. In 384-well format, the % CV for control cells was
11% and 3% for treated. The "z" scores were calculated as 0.74 in
96-well and 0.78 in the 384-well formats, confirming that these
assay conditions are suitable for HTS. These data are summarized in
Table 3.
High-Throughput Screening and Hit Selection
[0328] An 115,000 compound library was screened using the screening
protocol outlined in Table 2B. Each compound was added to a single
well to a final concentration of .about.2.2 .mu.M. The final DMSO
concentration in test and control wells was 0.13%. Each plate
included negative controls of 0.13% DMSO (n=16) and positive
controls of 500 mg/mL sodium butyrate with 0.13% DMSO (n=16). For
the entire screen, the average % CV was 9.1% for DMSO alone and
9.8% for sodium butyrate treated wells. The average increase with
500 mg/mL was 3.1 fold or 210%. A hit was defined by activation of
greater than 6 times the % CV; 60%. 462 hits were identified for an
overall 0.4% hit rate.
Hit Confirmation
[0329] The 462 hits were re-plated from the screening library into
master plates and then re-screened at 0.1, 1, and 5 M in
quadruplicate under conditions identical to the original HTS. Each
compound was counter-screened against an unrelated luciferase
control cell line that expresses luciferase from the minimal SV40
promoter and lacks an intron. This reporter should not respond to
compounds that specifically affect the SMN promoter or compounds
that change regulation of splicing. This allowed exclusion of
compounds that may cause non-specific increases in luciferase
expression. Of the 462 initial hits, 168 failed to reproduce at
least 60% activation with the SMN2-luciferase reporter clone and
were categorized as false positives (34% false positive rate). The
remaining 294 compounds were limited to a high priority group of 19
scaffolds based on potency, strength of activation, dose
dependency, specificity against luciferase control, and favorable
chemical properties. All 19 compounds showed greater than 100%
increase in reporter expression in the re-screen and stimulated the
control reporter less than 40%. All 19 lacked overtly toxic
functional groups and had chemical scaffolds that were tractable to
chemical modification.
[0330] Compounds then re-screened in quadruplicate using a
twelve-point dose response under the same conditions as the first
two rounds of screening with both the SMN2-luc and SMN1-luc cell
lines and using the SV40 luciferase control cell line. Since the
SMN1 and SMN2 promoters are nearly identical, compounds that
increased transcription of the SMN2-luc reporter would also
increase transcription of the SMN1-luc reporter. The SMN-luc cells
should therefore be responsive to compounds that increase
transcription from the SMN promoter. However, since the SMN gene in
the SMN1 cells includes exon 7 efficiently (>90%) only a small
increase in % activation is possible with compounds that stimulate
exon 7 inclusion.
[0331] Overall, this panel of reporter cell lines offers the
ability to discriminate between compounds that increase SMN
expression through promoter and splicing specific mechanisms and
rule out non-specific activators. For example LDN-109657 (FIG. 6B)
increased SMN2-luciferase levels up to 300% (4 fold), but also
increased luciferase expression in the SMN-luc1 reporter and, to a
lesser extent, the luciferase control cells. These data suggest
that this compound acts by either increasing SMN transcription or
protein stabilization. Several compounds decreased SMN1-luciferase
expressed in the SMN1-luc reporter cell line, but still increased
SMN2-luciferase expression in the SMN2-luc cell line (LDN-72939 and
LDN-79199 FIG. 6B). Of 17 compounds tested in the twelve-point dose
response, 13 increased luciferase expression by >60%, four did
not. Initial confirmation and characterization of three of these
compounds, LDN-72939, LDN-79199, and LDN-109657 is reported here.
Overall, the amplitude of SMN-luciferase increase varied, but all
three compounds displayed an EC50 in the low micromolar range; 1.1
.mu.M, 750 nM, and 2.4 .mu.M respectively.
[0332] To further evaluate the compounds, their effects on
endogenous SMN protein levels were examined. Any cell containing
even one copy of the SMN1 gene will produce enough SMN protein from
SMN1 to mask the effects of the compounds on protein expression
from the SMN2 gene. SMN null cells are not viable. The current
standard is to test primary human fibroblast cells derived from a
severe type 1 SMA patient. Most commonly used are the 3813 cells
(SMN1.sup.-/-; SMN2.sup.+/+). These cells express very low basal
levels of full-length SMN protein. For comparison, 3814 primary
fibroblasts from the carrier parent (SMN1.sup.+/-; SMN2.sup.+/+) of
this SMA infant are available. By quantitative immunoblot for
full-length SMN protein, the heterozygous 3814 cells express 3-5
times more full-length SMN protein than in the 3813 cells.sup.28
(FIG. 6C).
[0333] The selected compounds were tested for their effect on total
endogenous SMN protein levels. 3813 fibroblasts were treated with
varying concentrations of each compound for 72 hours (FIG. 6C). It
was observed that these primary fibroblasts were sensitive to lower
concentrations of compound than those used in the immortal reporter
cell lines. Not all compounds increase endogenous SMN levels in the
fibroblasts as well as others, regardless of their activity in the
reporter cell lines. LDN-72939 was more active in the reporter cell
line but showed little activity in the fibroblasts. LDN-79199 was
less active in the reporter cells, but increased endogenous SMN
level 2-fold at 370 nM. LDN-109657 displayed high activity in both
the reporter assay and in fibroblasts. It increased endogenous SMN
levels 2-fold at 120 nM. The decrease in SMN at higher
concentrations (1.1 M and 3.3 M) is likely due to either poor
solubility or a decrease in cell proliferation in the
fibroblasts.
Discussion
[0334] Spinal muscular atrophy is caused by an insufficient amount
of functional, full-length SMN protein, usually resulting from
deletion or disruption of both SMN1 alleles. All SMA patients
retain at least one SMN2 gene and disease severity inversely
correlates with SMN2 copy number. Since the SMN2 gene has the
potential to express functional full-length SMN protein, it is an
attractive therapeutic target for the treatment for SMA.
[0335] SMN2 expression can be increased through upregulation of
transcription, promotion of exon 7 inclusion, stabilization of SMN2
mRNA and protein, or elevation of translation efficiency. HDAC
inhibitors are known to influence transcription through histone
deacetylation, which causes chromatin remodeling. HDAC inhibitors
sodium butyrate, TSA, VPA, and SAHA all increased the amount of
total and exon 7 included SMN2-luc reporter transcripts (FIG. 4B).
Not only do these pan-HDAC inhibitors increase the quantity of
SMN-luciferase transcripts, but a higher percentage of those
transcripts are correctly spliced (FIG. 4B). This bimodal mechanism
for pan-HDAC inhibitor regulation of SMN2 expression may explain
the high potency and efficacy observed with these compounds, but
may also predict other potential and possibly detrimental
off-target effects.
[0336] Splicing of exon 7 is tightly regulated in both the SMN1 and
SMN2 mRNAs. Conserved splicing enhancer and splicing silencing
elements within exon 7 recruit splicing factors to the mRNA in
order to regulate exon 7 recognition. Two of these splicing
factors, hTra2.beta. and SF2/ASF, were tested in the SMN2-luc
reporter cell line. SF2/ASF had no impact on SMN2-luciferase
expression (FIGS. 5A-5C). hTra2.beta. binds to a splicing enhancer
downstream of the hnRNP A1 site and can stabilize U1 snRNP binding
at the 5'splice site of exon 7. This should increase exon 7
recognition and increase its splicing efficiency. An increase in
SMN-luciferase activity was observed, and an increase in exon 7
included full-length SMN2-luciferase transcripts with hTra2.beta.
overexpression (FIGS. 5A-5C). The small increase in total reporter
mRNA that accompanies this effect may be due to changes in the
stability of the full-length, exon 7 included mRNA and not an
actual increase in transcription of the reporter.
[0337] Disruption or regulation of the mRNA or protein turnover
machinery could also increase steady-state SMN levels. This could
be the mechanism for the activity of indoprofen, the
aminoglycosides, and the quinazoline compounds. Indoprofen, a
non-steroidal anti-inflammatory drug, was identified using a first
generation reporter assay. Its mechanism of action has not been
determined but recent evidence suggests that it has anti-terminator
activity that may stabilize the SMN A7 protein. It is possible that
the quinazoline compounds could increase the translation efficiency
of SMN mRNA, as suggested by the recent report of interaction with
the RNA decapping factor DcpS.
[0338] The SMN2 reporter cell line described herein combines the
benefits of previous reporters and expands their potential to
identify new regulatory circuitry for SMN2 expression. This assay
has proven to be stable and reliable in 96, 384, and 1536-well
formats and has been used in at three independent screening centers
to identify novel modulators of SMN2 protein expression.
Interestingly, the HTS at the NIH Chemical Genomics Center
identified chemically distinct activators. In addition to novel
compounds, these screens also identified compounds known to modify
SMN2 protein expression, including indoprofen, resveratrol,
hydroxyurea, and aclarubicin.
[0339] Characterization of LDN-72939, LDN-79199, and LDN-109657 are
shown herein. Each of these compounds increases SMN-luciferase
expression by at least 60%. LDN-79199 and LDN-109657 increased the
levels of endogenous SMN protein in SMA-derived primary
fibroblasts.
[0340] One of the drawbacks of the previous C33a reporter cell line
was its high basal level of SMN-luciferase expression. This made
detection of small increases in SMN-luciferase difficult. High
basal level expression was initially encountered with the new
HEK293 mixed-population reporter cells as well. By isolating and
expanding clonal cell lines, cells could be selected for lower
levels of basal SMN2-luciferase expression. Another issue with the
previous C33a reporter was a progressive loss of luciferase signal
intensity and decreased responsiveness to drug treatment. This
variability in signal strength and drug response made the previous
generation reporter unsuitable for high-throughput screening. The
new HEK293 cell lines have maintained constant luciferase
expression and reproducible induction with drug treatment over
hundreds of population doublings. The improvement in this reporter
assay is likely due to a combination of factors, including the new
reporter design, the episomal nature of the vector, isolation of
clonal cell lines, and use of HEK293 cells.
[0341] Spinal muscular atrophy is fatal for approximately 5,000
children each year in the United States, while tens of thousands of
others live with limited mobility and progressive muscle weakness.
There is presently no FDA approved drug for the treatment of spinal
muscular atrophy. It has been established that the amount of SMN
protein expressed has an inverse relationship to the severity to
the disease. Compounds that safely increase SMN protein levels
would dramatically improve the quality of life for individuals with
SMA. The assay described herein is a valuable tool for identifying
new compounds, characterizing existing compounds, and driving
medicinal chemistry programs on active chemical scaffolds.
TABLE-US-00002 TABLE 2a SMN-luciferase standard conditions - 96
well format Sequence Parameter Value Description 1 Cells 100 .mu.L
50,000 cells/well 96 TC treated white plate (See below for media
.sup.a) 2 Time 24 hours 37.degree. C. 5% CO.sub.2 3 Compound 100
.mu.L With compound 2x concentration 4 Time 24 hours 37.degree. C.
5% CO.sub.2 5 Remove media from wells 6 Reagent 30 .mu.L SteadyGlo
or DualGlo reagent (Promega) 7 Time 30 sec Room Temp 8 Detector See
below .sup.b Wallac Envision .sup.a Media: phenol red free DMEM +
10% FBS and 1x Pen/Strep .sup.b Wallac settings; luminescent read,
1.0 sec integration
TABLE-US-00003 TABLE 2b SMN2-luciferase conditions for HTS - 384
well format Sequence Parameter Value Description 1 Cells 40 .mu.L
10,000 cells/well 384 TC treated white plate (See below for media
.sup.c) 2 Time 1 hour 37.degree. C. 5% CO.sub.2 3 Compound 2.2 mM
Final of 0.13% DMSO 4 Time 25 hours 37.degree. C. 5% CO.sub.2 5
Reagent 25 .mu.L Perkin Elmer SteadyLite 6 Time 30 minutes Room
Temp 7 Detector See Below.sup.d LJL Analyst HT .sup.c Media:
Phenol-free DMEM + 10% FBSand 1x Pen/Strep .sup.dLJL Analyst HT
settings; luminescent read, 10 sec integration
TABLE-US-00004 TABLE 3 SMN2-luciferase Reporter validation with 0.5
mg/mL sodium butyrate 384- well LDDN Format 96-well validation
384-well validation screen Cells per well 25,000 50,000 25,000
50,000 5,000 7,500 10,000 10,000 Time point 24 hrs 24 hrs 48 hrs 48
hrs 24 hrs 24 hrs 24 hrs 24 hrs Na-But RLU* ave 2046 4798 3696 7505
1332 2832 39287 13894 Na-But stdev 138 234 536 756 131 329 1337
1272.55 CV Na-But 0.07 0.05 0.15 0.10 0.10 0.12 0.03 0.09 DMSO RLU*
ave 684 1357 1425 2626 521 1008 8054 4677 DMSO stdev 44 64 61.7 248
98 123 910 387 CV DMSO 0.06 0.05 0.04 0.09 0.19 0.12 0.11 0.08 S/B
3.0 3.5 2.6 2.9 2.6 2.8 4.9 3.0 Z' 0.60 0.74 0.21 0.38 0.15 0.26
0.78 0.46 *Relative Light Units (RLU) vary based on luciferase
substrate, plate reader, and protocol
TABLE-US-00005 TABLE 4 Cell-based reporter response to known SMN2
modulating compounds Max. fold increase EC.sub.50 Aclarubicin 10
298 nM Indoprofen 3 460 nM Sodium Butyrate 10 1 mg/mL Trichostatin
A 15 230 nM SAHA 15 12 .mu.M Valproic Acid 2.5 10 mM Tobramycin
inactive .sup.a inactive .sup.a .sup.a Transcriptional read-through
compounds do not score in this assay
TABLE-US-00006 TABLE 5 Activity of compounds tested in reporter
assay % activ- Structure LND # ation ##STR00164## 66854 210%
##STR00165## 66278 114% ##STR00166## 107992 121% ##STR00167## 32531
108% ##STR00168## 79199 102% ##STR00169## 110116 97% ##STR00170##
76070 122% ##STR00171## 79213 90% ##STR00172## 98541 124%
##STR00173## 110181 120% ##STR00174## 110425 93% ##STR00175## 67615
105% ##STR00176## 109981 98% ##STR00177## 75654 106% ##STR00178##
109657 152% ##STR00179## 75847 85% ##STR00180## 72939 102%
##STR00181## 75676 136%
TABLE-US-00007 TABLE 6 Additional compounds tested in the reporter
assay Ec50 Max % Compound Structure Active (.mu.M) Activity
LDN-75654 ##STR00182## Yes 1.8 274 LDN-75676 ##STR00183## Yes 2.6
337 LDN-75847 ##STR00184## Yes 9.9 333 LDN-75879 ##STR00185## Yes
1.5 180 CAL-1 ##STR00186## No -- -- CAL-2 ##STR00187## No -- --
CAL-3 ##STR00188## No -- -- CAL-4 ##STR00189## Yes 8 211 CAL-5
##STR00190## Yes 10 108 CAL-6 ##STR00191## Yes 6.5 180 CAL-7
##STR00192## Yes 10 134 CAL-8 ##STR00193## Weak 10 120 CAL-9
##STR00194## No -- -- CAL-10 ##STR00195## Yes 2 250 CAL-25
##STR00196## Yes 0.35 237 CAL-26 ##STR00197## Yes 94 160 CAL-27
##STR00198## Yes 1 125 CAL-28 ##STR00199## Yes 0.9 59 CAL-29
##STR00200## No -- -- CAL-30 ##STR00201## No -- -- CAL-31
##STR00202## Yes 16 154 CAL-33 ##STR00203## Yes 1.7 217 CAL-36
##STR00204## No -- -- CAL-37 ##STR00205## Yes 30 143 CAL-38
##STR00206## No -- -- CAL-42 ##STR00207## No -- -- LDN-211826
##STR00208## Yes 1.1 150 LDN-211827 ##STR00209## No/Weak -- --
LDN-211828 ##STR00210## Yes 20 100 LDN-211829 ##STR00211## No/Weak
-- -- LDN-211830 ##STR00212## No -- -- LDN-211831 ##STR00213## Yes
3 150 LDN-211832 ##STR00214## No -- -- LDN-211834 ##STR00215## No
-- -- LDN-211854 ##STR00216## Yes 0.5 200 LDN-211855 ##STR00217##
Yes 1.5 125 LDN-211856 ##STR00218## No -- -- LDN-211857
##STR00219## No -- -- LDN-211858 ##STR00220## No -- -- LDN-211859
##STR00221## No -- -- LDN-211860 ##STR00222## Yes 2.5 125
LDN-211861 ##STR00223## Yes 3.7 100 LDN-211862 ##STR00224##
Nonspecific -- -- LDN-211906 ##STR00225## Yes 0.8 250 LDN-211907
##STR00226## No/Weak -- -- LDN-211908 ##STR00227## No/Weak -- --
LDN-211909 ##STR00228## No/Weak -- -- LDN-211910 ##STR00229## Yes
0.7 100 LDN-211911 ##STR00230## Yes 0.4 175 LDN-211912 ##STR00231##
Yes 1.1 150 LDN-211981 ##STR00232## Yes 5.9 100 LDN-211982
##STR00233## No -- -- LDN-211983 ##STR00234## No -- -- LDN-211984
##STR00235## Yes 2.1 200 LDN-211985 ##STR00236## Yes 0.8 150
LDN-211986 ##STR00237## Yes 1.5 100 LDN-211992 ##STR00238## Yes
0.15 100 LDN-212011 ##STR00239## No -- -- LDN-212012 ##STR00240##
No -- -- LDN-212013 ##STR00241## Yes 0.5 100 LDN-212014
##STR00242## Yes 0.5 180 LDN-212015 ##STR00243## Yes 1.7 100
LDN-212016 ##STR00244## Yes 0.3 250 LDN-212026 ##STR00245## No --
-- LDN-212255 ##STR00246## Yes 0.4 117 LDN-212256 ##STR00247## Yes
0.4 114 LDN-213766 ##STR00248## No -- -- LDN-214095 ##STR00249## No
-- -- LDN-214098 ##STR00250## Yes 0.15 200 LDN-76070 ##STR00251##
Yes 6.6 198 LDN-76158 ##STR00252## No -- -- LDN-76515 ##STR00253##
No -- -- LDN-76074 ##STR00254## No/Weak -- -- CAL-21 ##STR00255##
Yes 251 14 CAL-22 ##STR00256## No -- -- CAL-23 ##STR00257## No --
-- CAL-50 ##STR00258## No -- -- CAL-51 ##STR00259## No -- -- CAL-52
##STR00260## No -- -- CAL-53 ##STR00261## Yes 91 27 CAL-55
##STR00262## No -- -- CAL-56 ##STR00263## No -- -- CAL-57
##STR00264## No -- -- LDN-212263 ##STR00265## No -- -- LDN-212264
##STR00266## No -- -- LDN-212265 ##STR00267## No -- -- LDN-212266
##STR00268## No/Weak -- -- LDN-212267 ##STR00269## No -- --
LDN-212350 ##STR00270## No/Weak -- -- LDN-212351 ##STR00271## Yes
12 150 LDN-212352 ##STR00272## No -- -- LDN-212353 ##STR00273##
No/Weak -- -- LDN-212354 ##STR00274## No/Weak -- -- LDN-212355
##STR00275## No/Weak -- -- LDN-212356 ##STR00276## Yes 43 150
LDN-212357 ##STR00277## No -- -- LDN-212358 ##STR00278## No -- --
LDN-212359 ##STR00279## No -- -- LDN-212360 ##STR00280## No -- --
LDN-212361 ##STR00281## No -- -- LDN-212388 ##STR00282## No -- --
LDN-212389 ##STR00283## No/Weak -- -- LDN-212390 ##STR00284## No --
-- LDN-212391 ##STR00285## Yes 4.4 267 LDN-212392 ##STR00286## No
-- -- LDN-212393 ##STR00287## No/Weak -- -- LDN-213767 ##STR00288##
Yes 8.4 125 LDN-213768 ##STR00289## Yes 5.9 150 LDN-213769
##STR00290## No -- -- LDN-213770 ##STR00291## No -- -- LDN-213771
##STR00292## No -- -- LDN-213772 ##STR00293## No -- -- LDN-213773
##STR00294## No -- -- LDN-214085 ##STR00295## Yes 6 180 LDN-214096
##STR00296## No -- -- LDN-214097 ##STR00297## No -- --
TABLE-US-00008 TABLE 7 Structure activity data Maximal Maximum %
T.sub.1/2 in liver solubility in activation EC.sub.50 microsomes
PBS 75654 242.4 .+-. 35.1 2.0 .mu.M .+-. 0.9 15 min >2 mM 212016
162.4 .+-. 62.4 0.3 .mu.M .+-. 0.1 Not tested 500 .mu.M 212014
156.6 .+-. 30.7 0.3 .mu.M .+-. 0.3 39 min >2 mM 76070 185.7 .+-.
48.0 8.3 .mu.M .+-. 4.5 Not tested 500 .mu.M 212351 168.5 .+-. 10.6
9.8 .mu.M .+-. 4.1 45 min 500 .mu.M 212391 238.3 .+-. 28.5 5.0
.mu.M .+-. 0.8 13 min 1 mM ##STR00298## ##STR00299## ##STR00300##
##STR00301## ##STR00302## ##STR00303## ##STR00304## ##STR00305##
##STR00306## ##STR00307##
Example 2
[0342] In this Example, compounds were tested using SMN2 reporter
cells. Particularly, over >150,000 compounds were tested using
SMN2 reporter cells. Secondary assays were performed to measure SMN
protein levels using SMA type 1 patient-derived fibroblasts (3813
cells) by Western blot, gem counts, and quantitative RT-PCR of SMN
transcripts.
[0343] Two of the compounds, LDN-75654 and LDN-76070, were further
tested. Analogs of both were synthesized and characterized first in
the SMN2-luc reporter assays and later by Western blot or gem count
using 3813 SMA fibroblasts (FIGS. 25A and 25B). Those compounds
that were validated and had tractable chemistry progressed to a
synthetic phase in which scaffolds are modified and their
properties evaluated. The compounds identified by this iterative
process are included in this application.
[0344] Seventy-six (76) LDN 75654 analogs were tested to explore
three different regions of the molecule, including introducing a
hydroxyl group to various aliphatic substituents on the central
heterocycle, replacing the isoxazole with other heterocycles (i.e.
thiazole and pyridine) and creating variations to the anilide
group. Exemplary compounds are shown below:
##STR00308## ##STR00309##
Exemplary analogs that emerged with activity are LDN-212016 (see
FIG. 25A), LDN-212014, LDN-212255, and LDN-212256. Some of the
compounds have different metabolic stability. For example,
LDN-212014 had a t.sub.1/2 of 39 minutes in mouse liver microsomes
compared to 14 min for LDN-75654.
Example 3
[0345] Forty eight (48) LDN-76070 analogs were evaluated. Exemplary
compounds are shown below:
##STR00310##
Modifications of the pendent phenyl were examined. In some
embodiments, the 2,5-dihalo substituted compounds showed activity.
The 2,5-dihalo substituted compounds had improved activity, such as
LDN-212351. The change of bromine to chlorine increased stability,
which may be advantageous. Not wishing to be limited by theory, the
increased stability could be due to C--Cl bonds being stronger than
C--Br bonds. LDN-212351 demonstrated a t.sub.1/2 of 45 min in mouse
liver microsomes. Incorporation of an additional nitrogen atom into
the central ring to generate urea LDN-212391 was tolerated (see
FIG. 25A).
[0346] Exemplary results are listed in Table 8, below.
TABLE-US-00009 TABLE 8 Exemplary compound test results. 75654
212014 212016 214098 76070 212351 212391 214085 +++ ++++ ++++ ++++
++ ++ +++ ++ Potency Luc Reporter ++++ +++ +++ +++ ++ ++ +++ ++
Activity Luc Reporter ++ ? ? ? ++++ +++ +++ ? Fibroblasts (n = 16)
(n = 4) (n = 4) - ++++ +++ ? ++++ +++ +++ ? IP (n = 16) (n = 4) (n
= 16) (n = 4) (n = 4) injections/Activity in Mice + + - + - - +/- ?
Solubility in aqueous solution 15 min 39 min 3 min 59 min ? 45 min
13-20 min ? Stabilityin liver microsomes
[0347] Pilot experiments with the LDN compounds were initiated in
SMA mice. Compounds LDN-75654 and LDN-76070 were tested in SMA mice
to evaluate SMN-inducing activity in vivo. The compounds were
injected intracerebroventricular (ICV) into SMA mice. Injections
were performed daily for 3 days, and animals were harvested on day
3. LDN-76070 increased SMN protein in muscle and liver.
Importantly, SMN was also significantly elevated in spinal cord and
brain (Table 9). LDN-75654 did not increase SMN protein levels in
these animals. Without the intention to be limited by theory, it is
proposed that this may be due to the instability observed in the
liver microsomal stability assay.
TABLE-US-00010 TABLE 9 Exemplary test results in SMA mice*. 75654
212014 212016 214058 76070 212351 212391 214085 +++ ++++ ++++ ++++
++ ++ +++ ++ Potency Luc Reporter ++++ +++ +++ +++ ++ ++ +++ ++
Activity Luc Reporter ++ ? ? ? ++ ? ? ? Fibroblasts - ++++ +++ ?
++++ +++ +++ ? IP (n = 16) (n = 4) (n = 16) (n = 4) (n = 4)
Injections + + - + - - +/- ? Solubility 15 min 39 min 3 min 59 min
? 45 min 13-20 min ? Stability *Neonatal SMN D7 mice (Smn-/-
SMN2+/+ SMN.DELTA.7+/+) were injected intraperitoneally twice daily
for 3 days with compound or DMSO starting at post-natal day 2.
Tissues were harvested and immunoblotted for SMN and actin.
Representative data from brain.
[0348] The animal studies included new derivatives of LDN-76070 and
LDN-75654. Mice were injected intraperitoneally once daily from
post-natal day 1-10. Weight increase, time to right onto four legs,
and extension of lifespan were chosen as parameters for efficacy.
Compounds LDN-212014, LDN-212016, LDN-212351, and LDN-212391 were
injected into a cohort of animals (3-4 each). Mice showed an
extension of lifespan. In the Kaplan-Meier curve that DMSO control
decreased survival by 50%--the compounds were in 20% DMSO (FIG.
26A). The weight of the treated animals also increased and some of
the animals were able to right themselves starting at day 6 (FIG.
26B and Table 10).
TABLE-US-00011 TABLE 10 Time to right in seconds. Fail = >30 sec
(raw data). Pup Inj. DOB PND6 PND7 PND8 PND9 PND10 PND11 PND12
4527.5 212014 Apr. 5, 2011 Fail Fail Fail Fail Fail 5 16 4389.4
212014 Apr. 5, 2011 dead dead dead dead dead dead dead 4378.4
212014 Apr. 5, 2011 Fail Fail Fail 26 Fail Fail dead 4514.3 212014
Apr. 5, 2011 Fail Fail Fail Fail Fail Fail dead 4527.7 212016 Apr.
5, 2011 Fail Fail Fail Fail Fail Fail Fail 4389.1 212016 Apr. 5,
2011 Fail Fail Fail Fail Fail Fail Fail 4378.5 212016 Apr. 5, 2011
Fail 25 Fail Fail Fail Fail Fail 4515.5 212016 Apr. 5, 2011 Fail
Fail Fail Fail 12 9 Fail 4516.3 212351 Apr. 5, 2011 dead dead dead
dead dead dead dead 4533.5 212351 Apr. 5, 2011 Fail Fail Fail 13
Fail dead dead 4358.4 212351 Apr. 5, 2011 Fail Fail Fail Fail Fail
dead dead 4354.1 212351 Apr. 5, 2011 3 5 Fail Fail Fail Fail Fail
4516.4 212391 Apr. 5, 2011 Fail Fail Fail Fail Fail Fail dead
4514.2 212391 Apr. 5, 2011 Fail Fail Fail Fail Fail Fail Fail
4354.4 212391 Apr. 5, 2011 Fail Fail Fail 18 20 Fail Fail 4378.2
DMSO Apr. 5, 2011 Fail Fail Fail Fail dead dead dead 4258.6 DMSO
Apr. 5, 2011 dead dead dead dead dead dead dead 4514.1 DMSO Apr. 5,
2011 dead dead dead dead dead dead dead 4515.6 DMSO Apr. 5, 2011
dead dead dead dead dead dead dead Pup PND13 PND14 PND15 PND16
PND17 PND18 PND19 PND20 4527.5 Fail 5 14 Fail Fail dead dead dead
4389.4 dead dead dead dead dead dead dead dead 4378.4 dead dead
dead dead dead dead dead dead 4514.3 dead dead dead dead dead dead
dead dead 4527.7 dead dead dead dead dead dead dead dead 4389.1
Fail dead dead dead dead dead dead dead 4378.5 Fail Fail dead dead
dead dead dead dead 4515.5 11 Fail Fail Fail Fail dead dead dead
4516.3 dead dead dead dead dead dead dead dead 4533.5 dead dead
dead dead dead dead dead dead 4358.4 dead dead dead dead dead dead
dead dead 4354.1 Fail Fail dead dead dead dead dead dead 4516.4
dead dead dead dead dead dead dead dead 4514.2 Fail Fail Fail Fail
Fail Fail Fail dead 4354.4 Fail Fail Fail dead dead dead dead dead
4378.2 dead dead dead dead dead dead dead dead 4258.6 dead dead
dead dead dead dead dead dead 4514.1 dead dead dead dead dead dead
dead dead 4515.6 dead dead dead dead dead dead dead dead
[0349] LDN-212014 and LDN-76070 were further tested in A7 SMA model
mice. Mice were dosed using compounds dissolved in 100% DMSO
injected at 20 mg/kg with each dose administered once daily by
intraperitoneal injection. As shown in the black curves in the
Kaplan-Meier plot, control animals lived 4-5 days longer than the
DMSO vehicle treated animals, indicating toxicity. Nonetheless,
both 76070 and 212014 compounds significantly increased lifespan
despite this DMSO associated toxicity (p<0.0001). In fact, the
compounds display a statistically significant increase in survival
compared with the untreated animals. These compounds extend the
lifespan of SMA mice despite the DMSO toxicity. Test results of
LDN-212014 and LDN-76070 were shown in FIGS. 8-24, 27-28 and
others.
[0350] These experiments also demonstrated there was a concomitant
increase in motor function in the treated animals. In `time to
right` experiments, animals are placed on their backs and the
duration for animals to right themselves is recorded. Animals that
are unable to right in less than 30 seconds are scored as a
failure. DMSO treated animals were unable to right themselves,
while a small percentage of untreated animals display the ability
to right themselves for up to postnatal day (PND) 11. A larger
percentage of treated animals can right themselves past PND 17, and
in the case of one animal as late as PND 21.
[0351] To assess SMN protein level, tissues were harvested from
three treated animals from each experiment at PND 7 and compared to
wild type, untreated, and DMSO treated animals. Tissues from brain,
spinal cord, and muscle were assayed. As shown in the western blots
(FIG. 29), both compounds increase SMN protein levels in brain and
spinal cord to greater than 75% that of wild type animals. They
also promoted an increase of about 25% that of wild type in muscle.
High levels of increase in these tissues are very desirable,
especially in tissues of the CNS, suggesting that these compounds
can pass through the blood brain barrier.
Exemplary Synthesis of Compounds.
[0352] In some embodiments, certain compounds described in the
application are prepared using the general procedure outlined in
Scheme 1 (Synthesis 2006, (22), 3805; Jiegou Huaxue 2007, 26(5),
533; Tetrahedron Lett. 2005, 46, 7169):
##STR00311##
In some embodiments, several of the urea analogs are prepared using
the procedures outlined in Scheme 2 (Perkins Trans 2 2001, 2226 and
J. Heterocycl Chem 1982, 19, 1453):
##STR00312##
In other embodiments, several seven-member ring analogs are
prepared using the procedures outlined in Scheme 3 (J. Am. Chem.
Soc. 1954, 76, 4550; J. Med Chem. 1974, 17, 668; J. Med Chem. 2006,
49, 3520-3535; J. Org. Chem. 1986, 51, 5001-5002):
##STR00313## ##STR00314##
[0353] In some embodiments, certain compounds are prepared using
the procedure outline in Scheme 4 with aryl or heteroaryl acyl
chlorides and aryl or heteroaryl amines. In other embodiments, a
two-step process is used starting with aryl or heteroaryl
carboxylic acids.
##STR00315##
In some embodiments, certain heteroaryl carboxylic acids are
prepared using the procedure outlined in Scheme 5 (Eur. J. Org.
Chem. 2007, 4352-4359) and in Scheme 6 (J. Am. Chem. Soc. 1992,
114, 9450 and J. Org. Chem. 1993, 58, 4495).
##STR00316##
##STR00317##
In some embodiments, the cyclohexyl analog is prepared using the
procedure outlined in Scheme 7 (Synthesis 1992, 769).
##STR00318##
In some embodiments, certain heteroaryl amines are prepared using
the procedure outlined in Scheme 8.
##STR00319##
Sequence CWU 1
1
9125DNAArtificial SequenceSynthetic 1ccacaaatgt gggagggcga taacc
25235DNAArtificial SequenceSynthetic 2tatctcgagt ggtccagaag
gaaatggagg cagcc 35323DNAArtificial SequenceSynthetic 3catttccttc
tggaccactc gag 23422DNAArtificial SequenceSynthetic 4atagcttctg
ccaaccgaac gg 22524DNAArtificial SequenceSynthetic 5taaggaatgt
gagcaccttc cttc 24620DNAArtificial SequenceSynthetic 6tccaccaccc
tgttgctgta 20720DNAArtificial SequenceSynthetic 7accacagtcc
atgccatcac 20820198DNAArtificial SequenceSynthetic 8gttgacattg
attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat
ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
acgccaatag 180ggactttcca ttgacgtcaa tgggtggagt atttacggta
aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtccgcccc
ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac
atgaccttac gggactttcc tacttggcag tacatctacg 360tattagtcat
cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat
420agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
gggagtttgt 480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
taaccccgcc ccgttgacgc 540aaatgggcgg taggcgtgta cggtgggagg
tctatataag cagagctcgt ttagtgaacc 600gtcagatctc tagaagctgg
gtaccagctg ctagccacca tggcttccaa ggtgtacgac 660cccgagcaac
gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg
720aacgtgctgg actccttcat caactactat gattccgaga agcacgccga
gaacgccgtg 780atttttctgc atggtaacgc tgcctccagc tacctgtgga
ggcacgtcgt gcctcacatc 840gagcccgtgg ctagatgcat catccctgat
ctgatcggaa tgggtaagtc cggcaagagc 900gggaatggct catatcgcct
cctggatcac tacaagtacc tcaccgcttg gttcgagctg 960ctgaaccttc
caaagaaaat catctttgtg ggccacgact ggggggcttg tctggccttt
1020cactactcct acgagcacca agacaagatc aaggccatcg tccatgctga
gagtgtcgtg 1080gacgtgatcg agtcctggga cgagtggcct gacatcgagg
aggatatcgc cctgatcaag 1140agcgaagagg gcgagaaaat ggtgcttgag
aataacttct tcgtcgagac catgctccca 1200agcaagatca tgcggaaact
ggagcctgag gagttcgctg cctacctgga gccattcaag 1260gagaagggcg
aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag
1320ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct
tcgggccagc 1380gacgatctgc ctaagatgtt catcgagtcc gaccctgggt
tcttttccaa cgctattgtc 1440gagggagcta agaagttccc taacaccgag
ttcgtgaagg tgaagggcct ccacttcagc 1500caggaggacg ctccagatga
aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg 1560aagaacgagc
agtaattcta gagcggccgc tcgaggccgg caaggccgga tccagacatg
1620ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
aaaatgcttt 1680atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
ttataagctg caataaacaa 1740gttaacaaca acaattgcat tcattttatg
tttcaggttc agggggaggt gtgggaggtt 1800ttttaaagca agtaaaacct
ctacaaatgt ggtatggctg attatgatcc ggctgcctcg 1860cgcgtttcgg
tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
1920cttgtctgta agcggatgcc gggagcagac aagcccgtca ggcgtcagcg
ggtgttggcg 1980ggtgtcgggg cgcagccatg aggtcgactc tagaggatcg
atgccccgcc ccggacgaac 2040taaacctgac tacgacatct ctgccccttc
ttcgcggggc agtgcatgta atcccttcag 2100ttggttggta caacttgcca
actgggccct gttccacatg tgacacgggg ggggaccaaa 2160cacaaagggg
ttctctgact gtagttgaca tccttataaa tggatgtgca catttgccaa
2220cactgagtgg ctttcatcct ggagcagact ttgcagtctg tggactgcaa
cacaacattg 2280cctttatgtg taactcttgg ctgaagctct tacaccaatg
ctgggggaca tgtacctccc 2340aggggcccag gaagactacg ggaggctaca
ccaacgtcaa tcagaggggc ctgtgtagct 2400accgataagc ggaccctcaa
gagggcatta gcaatagtgt ttataaggcc cccttgttaa 2460ccctaaacgg
gtagcatatg cttcccgggt agtagtatat actatccaga ctaaccctaa
2520ttcaatagca tatgttaccc aacgggaagc atatgctatc gaattagggt
tagtaaaagg 2580gtcctaagga acagcgatat ctcccacccc atgagctgtc
acggttttat ttacatgggg 2640tcaggattcc acgagggtag tgaaccattt
tagtcacaag ggcagtggct gaagatcaag 2700gagcgggcag tgaactctcc
tgaatcttcg cctgcttctt cattctcctt cgtttagcta 2760atagaataac
tgctgagttg tgaacagtaa ggtgtatgtg aggtgctcga aaacaaggtt
2820tcaggtgacg cccccagaat aaaatttgga cggggggttc agtggtggca
ttgtgctatg 2880acaccaatat aaccctcaca aaccccttgg gcaataaata
ctagtgtagg aatgaaacat 2940tctgaatatc tttaacaata gaaatccatg
gggtggggac aagccgtaaa gactggatgt 3000ccatctcaca cgaatttatg
gctatgggca acacataatc ctagtgcaat atgatactgg 3060ggttattaag
atgtgtccca ggcagggacc aagacaggtg aaccatgttg ttacactcta
3120tttgtaacaa ggggaaagag agtggacgcc gacagcagcg gactccactg
gttgtctcta 3180acacccccga aaattaaacg gggctccacg ccaatggggc
ccataaacaa agacaagtgg 3240ccactctttt ttttgaaatt gtggagtggg
ggcacgcgtc agcccccaca cgccgccctg 3300cggttttgga ctgtaaaata
agggtgtaat aacttggctg attgtaaccc cgctaaccac 3360tgcggtcaaa
ccacttgccc acaaaaccac taatggcacc ccggggaata cctgcataag
3420taggtgggcg ggccaagata ggggcgcgat tgctgcgatc tggaggacaa
attacacaca 3480cttgcgcctg agcgccaagc acagggttgt tggtcctcat
attcacgagg tcgctgagag 3540cacggtgggc taatgttgcc atgggtagca
tatactaccc aaatatctgg atagcatatg 3600ctatcctaat ctatatctgg
gtagcatagg ctatcctaat ctatatctgg gtagcatatg 3660ctatcctaat
ctatatctgg gtagtatatg ctatcctaat ttatatctgg gtagcatagg
3720ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg
gtagtatatg 3780ctatcctaat ctgtatccgg gtagcatatg ctatcctaat
agagattagg gtagtatatg 3840ctatcctaat ttatatctgg gtagcatata
ctacccaaat atctggatag catatgctat 3900cctaatctat atctgggtag
catatgctat cctaatctat atctgggtag cataggctat 3960cctaatctat
atctgggtag catatgctat cctaatctat atctgggtag tatatgctat
4020cctaatttat atctgggtag cataggctat cctaatctat atctgggtag
catatgctat 4080cctaatctat atctgggtag tatatgctat cctaatctgt
atccgggtag catatgctat 4140cctcatgcat atacagtcag catatgatac
ccagtagtag agtgggagtg ctatcctttg 4200catatgccgc cacctcccaa
gggggcgtga attttcgctg cttgtccttt tcctgctggt 4260tgctcccatt
cttaggtgaa tttaaggagg ccaggctaaa gccgtcgcat gtctgattgc
4320tcaccaggta aatgtcgcta atgttttcca acgcgagaag gtgttgagcg
cggagctgag 4380tgacgtgaca acatgggtat gcccaattgc cccatgttgg
gaggacgaaa atggtgacaa 4440gacagatggc cagaaataca ccaacagcac
gcatgatgtc tactggggat ttattcttta 4500gtgcggggga atacacggct
tttaatacga ttgagggcgt ctcctaacaa gttacatcac 4560tcctgccctt
cctcaccctc atctccatca cctccttcat ctccgtcatc tccgtcatca
4620ccctccgcgg cagccccttc caccataggt ggaaaccagg gaggcaaatc
tactccatcg 4680tcaaagctgc acacagtcac cctgatattg caggtaggag
cgggctttgt cataacaagg 4740tccttaatcg catccttcaa aacctcagca
aatatatgag tttgtaaaaa gaccatgaaa 4800taacagacaa tggactccct
tagcgggcca ggttgtgggc cgggtccagg ggccattcca 4860aaggggagac
gactcaatgg tgtaagacga cattgtggaa tagcaagggc agttcctcgc
4920cttaggttgt aaagggaggt cttactacct ccatatacga acacaccggc
gacccaagtt 4980ccttcgtcgg tagtcctttc tacgtgactc ctagccagga
gagctcttaa accttctgca 5040atgttctcaa atttcgggtt ggaacctcct
tgaccacgat gcttttccaa accaccctcc 5100ttttttgcgc cctgcctcca
tcaccctgac cccggggtcc agtgcttggg ccttctcctg 5160ggtcatctgc
ggggccctgc tctatcgctc ccgggggcac gtcaggctca ccatctgggc
5220caccttcttg gtggtattca aaataatcgg cttcccctac agggtggaaa
aatggccttc 5280tacctggagg gggcctgcgc ggtggagacc cggatgatga
tgactgacta ctgggactcc 5340tgggcctctt ttctccacgt ccacgacctc
tccccctggc tctttcacga cttccccccc 5400tggctctttc acgtcctcta
ccccggcggc ctccactacc tcctcgaccc cggcctccac 5460tacctcctcg
accccggcct ccactgcctc ctcgaccccg gcctccacct cctgctcctg
5520cccctcctgc tcctgcccct cctcctgctc ctgcccctcc tgcccctcct
gctcctgccc 5580ctcctgcccc tcctgctcct gcccctcctg cccctcctgc
tcctgcccct cctgcccctc 5640ctcctgctcc tgcccctcct gcccctcctc
ctgctcctgc ccctcctgcc cctcctgctc 5700ctgcccctcc tgcccctcct
gctcctgccc ctcctgcccc tcctgctcct gcccctcctg 5760ctcctgcccc
tcctgctcct gcccctcctg ctcctgcccc tcctgcccct cctgcccctc
5820ctcctgctcc tgcccctcct gctcctgccc ctcctgcccc tcctgcccct
cctgctcctg 5880cccctcctcc tgctcctgcc cctcctgccc ctcctgcccc
tcctcctgct cctgcccctc 5940ctgcccctcc tcctgctcct gcccctcctc
ctgctcctgc ccctcctgcc cctcctgccc 6000ctcctcctgc tcctgcccct
cctgcccctc ctcctgctcc tgcccctcct cctgctcctg 6060cccctcctgc
ccctcctgcc cctcctcctg ctcctgcccc tcctcctgct cctgcccctc
6120ctgcccctcc tgcccctcct gcccctcctc ctgctcctgc ccctcctcct
gctcctgccc 6180ctcctgctcc tgcccctccc gctcctgctc ctgctcctgt
tccaccgtgg gtccctttgc 6240agccaatgca acttggacgt ttttggggtc
tccggacacc atctctatgt cttggccctg 6300atcctgagcc gcccggggct
cctggtcttc cgcctcctcg tcctcgtcct cttccccgtc 6360ctcgtccatg
gttatcaccc cctcttcttt gaggtccact gccgccggag ccttctggtc
6420cagatgtgtc tcccttctct cctaggccat ttccaggtcc tgtacctggc
ccctcgtcag 6480acatgattca cactaaaaga gatcaataga catctttatt
agacgacgct cagtgaatac 6540agggagtgca gactcctgcc ccctccaaca
gcccccccac cctcatcccc ttcatggtcg 6600ctgtcagaca gatccaggtc
tgaaaattcc ccatcctccg aaccatcctc gtcctcatca 6660ccaattactc
gcagcccgga aaactcccgc tgaacatcct caagatttgc gtcctgagcc
6720tcaagccagg cctcaaattc ctcgtccccc tttttgctgg acggtaggga
tggggattct 6780cgggacccct cctcttcctc ttcaaggtca ccagacagag
atgctactgg ggcaacggaa 6840gaaaagctgg gtgcggcctg tgaggatcag
cttatcgatg ataagctgtc aaacatgaga 6900attcttgaag acgaaagggc
ctcgtgatac gcctattttt ataggttaat gtcatgataa 6960taatggtttc
ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
7020gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
ccctgataaa 7080tgcttcaata atattgaaaa aggaagagta tgagtattca
acatttccgt gtcgccctta 7140ttcccttttt tgcggcattt tgccttcctg
tttttgctca cccagaaacg ctggtgaaag 7200taaaagatgc tgaagatcag
ttgggtgcac gagtgggtta catcgaactg gatctcaaca 7260gcggtaagat
ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
7320aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
caactcggtc 7380gccgcataca ctattctcag aatgacttgg ttgagtactc
accagtcaca gaaaagcatc 7440ttacggatgg catgacagta agagaattat
gcagtgctgc cataaccatg agtgataaca 7500ctgcggccaa cttacttctg
acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 7560acaacatggg
ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
7620taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
ttgcgcaaac 7680tattaactgg cgaactactt actctagctt cccggcaaca
attaatagac tggatggagg 7740cggataaagt tgcaggacca cttctgcgct
cggcccttcc ggctggctgg tttattgctg 7800ataaatctgg agccggtgag
cgtgggtctc gcggtatcat tgcagcactg gggccagatg 7860gtaagccctc
ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
7920gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
ctgtcagacc 7980aagtttactc atatatactt tagattgatt taaaacttca
tttttaattt aaaaggatct 8040aggtgaagat cctttttgat aatctcatga
ccaaaatccc ttaacgtgag ttttcgttcc 8100actgagcgtc agaccccgta
gaaaagatca aaggatcttc ttgagatcct ttttttctgc 8160gcgtaatctg
ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
8220atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
cagataccaa 8280atactgtcct tctagtgtag ccgtagttag gccaccactt
caagaactct gtagcaccgc 8340ctacatacct cgctctgcta atcctgttac
cagtggctgc tgccagtggc gataagtcgt 8400gtcttaccgg gttggactca
agacgatagt taccggataa ggcgcagcgg tcgggctgaa 8460cggggggttc
gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
8520tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
gacaggtatc 8580cggtaagcgg cagggtcgga acaggagagc gcacgaggga
gcttccaggg ggaaacgcct 8640ggtatcttta tagtcctgtc gggtttcgcc
acctctgact tgagcgtcga tttttgtgat 8700gctcgtcagg ggggcggagc
ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 8760tggccttttg
ctggccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
8820tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata
atggggaagg 8880ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc
cagcgcgtcg gccccgagat 8940gcgccgcgtg cggctgctgg agatggcgga
cgcgatggat atgttctgcc aagggttggt 9000ttgcgcattc acagttctcc
gcaagaattg attggctcca attcttggag tggtgaatcc 9060gttagcgagg
tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg
9120tccccggaag aaatatattt gcatgtcttt agttctatga tgacacaaac
cccgcccagc 9180gtcttgtcat tggcgaattc gaacacgcag atgcagtcgg
ggcggcgcgg tccgaggtcc 9240acttcgcata ttaaggtgac gcgtgtggcc
tcgaacaccg agcgaccctg cagcgacccg 9300cttaacagcg tcaacagcgt
gccgcagatc ccggggggca atgagatatg aaaaagcctg 9360aactcaccgc
gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
9420tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
ggagggcgtg 9480gatatgtcct gcgggtaaat agctgcgccg atggtttcta
caaagatcgt tatgtttatc 9540ggcactttgc atcggccgcg ctcccgattc
cggaagtgct tgacattggg gaattcagcg 9600agagcctgac ctattgcatc
tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 9660aaaccgaact
gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
9720ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
ggtcaataca 9780ctacatggcg tgatttcata tgcgcgattg ctgatcccca
tgtgtatcac tggcaaactg 9840tgatggacga caccgtcagt gcgtccgtcg
cgcaggctct cgatgagctg atgctttggg 9900ccgaggactg ccccgaagtc
cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 9960tgacggacaa
tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
10020cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
atggagcagc 10080agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
atcgccgcgg ctccgggcgt 10140atatgctccg cattggtctt gaccaactct
atcagagctt ggttgacggc aatttcgatg 10200atgcagcttg ggcgcagggt
cgatgcgacg caatcgtccg atccggagcc gggactgtcg 10260ggcgtacaca
aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
10320tcgccgatag tggaaaccga cgccccagca ctcgtccgga tcgggagatg
ggggaggcta 10380actgaaacac ggaaggagac aataccggaa ggaacccgcg
ctatgacggc aataaaaaga 10440cagaataaaa cgcacgggtg ttgggtcgtt
tgttcataaa cgcggggttc ggtcccaggg 10500ctggcactct gtcgataccc
caccgagacc ccattggggc caatacgccc gcgtttcttc 10560cttttcccca
ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
10620ggcggcaggc cctgccatag ccactggccc cgtgggttag ggacggggtc
ccccatgggg 10680aatggtttat ggttcgtggg ggttattatt ttgggcgttg
cgtggggtca ggtccacgac 10740tggactgagc agacagaccc atggtttttg
gatggcctgg gcatggaccg catgtactgg 10800cgcgacacga acaccgggcg
tctgtggctg ccaaacaccc ccgaccccca aaaaccaccg 10860cgcggatttc
tggcgtgcca agctagtcga ccaattctca tgtttgacag cttatcatcg
10920cagatccggg caacgttgtt gccattgctg caggcgcaga actggtaggt
atggaagatc 10980tatacattga atcaatattg gcaattagcc atattagtca
ttggttatat agcataaatc 11040aatattggct attggccatt gcatacgttg
tatctatatc ataatatgta cctaaccaag 11100ttcctctttc agaggttatt
tcaggccatg gtgctgcgca gatccgcgta tgcggtgtga 11160aataccgcac
agatgcgtaa ggagaaaata ccgcatcagg cgaaattgta aacgttaata
11220ttttgttaaa attcgcgtta aatatttgtt aaatcagctc attttttaac
caataggccg 11280aaatcggcaa aatcccttat aaatcaaaag aatagaccga
gatagggttg agtgttgttc 11340cagtttggaa caagagtcca ctattaaaga
acgtggactc caacgtcaaa gggcgaaaaa 11400ccgtctatca gggcgatggc
ccactacgtg aaccatcacc caaatcaagt tttttgcggt 11460cgaggtgccg
taaagctcta aatcggaacc ctaaagggag cccccgattt agagcttgac
11520ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
gcgggcgcta 11580gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
cacacccgcc gcgcttaatg 11640cgccgctaca gggcgcgtcc attcgccatt
caggctgcgc aactgttggg aagggcgatc 11700ggtgcgggcc tcttcgctat
tacgccagcc cggatcgatc cttatcggat tttaccacat 11760ttgtagaggt
tttacttgct ttaaaaaacc tcccacatct ccccctgaac ctgaaacata
11820aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
tacaaataaa 11880gcaatagcat cacaaatttc acaaataaag catttttttc
actgcattct agttgtggtt 11940tgtccaaact catcaatgta tcttatcatg
tctgctcgaa gcattaaccc tcactaaagg 12000gaagcggccg cttacatttt
acaatttgga ctttccgccc ttcttggcct ttatgaggat 12060ctctctgatt
tttcttgcgt cgagttttcc ggtaagacct ttcggtactt cgtccacaaa
12120cacaactcct ccgcgcaact ttttcgcggt tgttacttga ctggcgacgt
aatccacgat 12180ctctttttcc gtcatcgtct ttccgtgctc caaaacaaca
acggcggcgg gaagttcacc 12240ggcgtcatcg tcgggaagac ctgccacgcc
cgcgtcgaag atgttggggt gttgtaacaa 12300tatcgattcc aattcagcgg
gggccacctg atatcctttg tatttaatta aagacttcaa 12360gcggtcaact
atgaagaagt gttcgtcttc gtcccagtaa gctatgtctc cagaatgtag
12420ccatccatcc ttgtcaatca aggcgttggt cgcttccgga ttgtttacat
aaccggacat 12480aatcataggt cctctgacac ataattcgcc tctctgatta
acgcccagcg ttttcccggt 12540atccagatcc acaaccttcg cttcaaaaaa
tggaacaact ttaccgaccg cgcccggttt 12600atcatccccc tcgggtgtaa
tcagaatagc tgatgtagtc tcagtgagcc catatccttg 12660tcgtatccct
ggaagatgga agcgttttgc aaccgcttcc ccgacttctt tcgaaagagg
12720tgcgccccca gaagcaattt cgtgtaaatt agataaatcg tatttgtcaa
tcagagtgct 12780tttggcgaag aatgaaaata gggttggtac tagcaacgca
ctttgaattt tgtaatcctg 12840aagggatcgt aaaaacagct cttcttcaaa
tctatacatt aagacgactc gaaatccaca 12900tatcaaatat ccgagtgtag
taaacattcc aaaaccgtga tggaatggaa caacacttaa 12960aatcgcagta
tccggaatga tttgattgcc aaaaatagga tctctggcat gcgagaatct
13020gacgcaggca gttctatgcg gaagggccac acccttaggt aacccagtag
atccagagga 13080attcattatc agtgcaattg ttttgtcacg atcaaaggac
tctggtacaa aatcgtattc 13140attaaaaccg ggaggtagat gagatgtgac
gaacgtgtac atcgactgaa atccctggta 13200atccgtttta gaatccatga
taataatttt ctggattatt ggtaattttt tttgcacgtt 13260caaaattttt
tgcaacccct ttttggaaac aaacactacg gtaggctgcg aaatgttcat
13320actgttgagc aattcacgtt cattataaat gtcgttcgcg ggcgcaactg
caactccgat 13380aaataacgcg cccaacaccg gcataaagaa ttgaagagag
ttttcactgc atacgacgat 13440tctgtgattt gtattcagcc catatcgttt
catagcttct gccaaccgaa cggacatttc 13500gaagtattcc gcgtacgtga
tgttcacctc gatatgtgca tctgtaaaag caattgttcc 13560aggaaccagg
gcgtatctct tcatagcctt atgcagttgc tctccagcgg ttccatcctc
13620tagaggatag aatggcgccg ggcctttctt tatgtttttg gcgtcttcca
gctgctctat 13680gccagcattt cctgcaaatg agaaattaga accagaggct
tgacgaattc cagttaaacc 13740atgtcctctg tggacaccag ttaaacttga
ctagagcact tcatatgtca gagtgtacag 13800tgcagtatgc ctaggttatc
ccatatcaca ataaaaaaaa gtctgctggt ctgcctacta 13860gtgatataaa
atggcatcat atcctaaagc tctttattgt gaaagtatgt ttcttccaca
13920taaccaacca gttaagtatg agaattctag tagggatgta gattaacctt
ttatctaata 13980gttttggcat caaaattctt taatattgat tgttttacat
taacctttca actttttaac 14040atctgaactt tttaaatgtt caaaaacatt
tgttttccac aaaccataaa gttttacaaa 14100agtaagattc actttcataa
tgctggcaga cttactcctt agatttaagg aatgtgagca 14160ccttccttct
ttttgatttt gtctgaaacc ctgtaaggaa aataaaggaa gttaaaaaaa
14220atagctatat agacatagat agctatatat agatagcttt atatggatgt
taaaaagcat 14280tttgtttcac aagacatttt acttatttta ttcaacaaaa
tatgatcaga aattaagttg 14340atagtctttt aatgtacttt aaaagttatc
ccaaagaaaa caattattag gctgcagtta
14400aggttttctt gcagtggctc atgcctacaa tcccacaact ttgggaggcg
gaggtagggg 14460gatcacttga gacctggagc ttgacaccac cctgggcaac
ataatgagac cctgtctcta 14520caaaaaattt aaaaattagg ccggcgtggt
ggctcaggct aggcacagtg gctcacgcct 14580gtaatcccag cactttggga
ggccgagaca gttggatcac ctgagctcag gagttcgaga 14640acagcctggc
caacatggca aaaccccatt tctactgaaa gtacaaaaaa ttagccaggc
14700atggtggtgg ggacctctaa tcccagctac ttgggaggct gaggcaggag
aatcacttga 14760acccaggagg cggaggctgc agtgagctga gatttacacc
actgcactcc agcctgggtg 14820acagagcaag actctgtctc aaaaaaaaat
aaataaataa aaataaaaat tagccaggtg 14880cagtggcatt atcactgtag
tcccagctac tcgggaaact gaggtgagag gactgcttga 14940gccctggagg
tcaaggctgc agtgagctgt gaatgtgccc ttgcactcca gcctgagcaa
15000tagagtgaga cctggtctct aaaaaataaa atttgggagg cggagcttgc
agtgagccga 15060gactgcgcca ctgtactcca gcctgggtga cagagcgaga
ctccgtctca aaaaaaaaaa 15120aaaaataaaa taaaataaaa taattttaaa
tgttctgact aaaatacaat agaacatgtc 15180cgtaggagac taacgtataa
agtgacaagt ttgaagccat actccccaag gttcaatgtg 15240gtacacatta
ccccagatct ttgtgcatta aaaaaatttc atttctcttg gaaggccgag
15300gcgggtggat cacggggtca ggagattgag accatcctgg ctaacacagt
gaaaccctgt 15360ctttacaaaa aaatacaaaa aattagacag gcgtggtggc
aggcacctgt agtcccagct 15420acctctgagg ctgaggcagg agaatggcgt
gaatccagaa ggcagagctt gctgtgagcc 15480aagatcacgc cattgcactc
cagcctgggc aacagagcaa gactccgtct caaaaaaaaa 15540aaaaaaaaaa
aaagaatttc atttttcatt tatgaaaaat tatcccatct tttccattcc
15600ctacaatcaa tttcaaatca gagattaaaa cattatttag aaaaagtata
atttcaattc 15660aaaagtgtat aatcaaaata atctaacaat agcatgaaag
ctttttaaaa ttaactaaaa 15720ttatacttag ggacaatgca agagtaattt
aagcctcaga cagttgtatt tttttatttt 15780tattttttag taatataaag
agagaagcaa gtagtatttt ataaatttac aaaacaaagt 15840cacataacta
caaaaaaatt gtcaggaaaa gatgctgagt gattacttac catataatag
15900ccagtatgat agccactcat gtaccatgaa attaacatac ttcccaaagc
atcagcatca 15960tcaagagaat ctggacatat gggaggtggt gggggaatta
tctcgagtgg tccagaagga 16020aatggaggca gccagcatga tagtaagtgg
ggtggtggtg gtggcggtgg cggtggtggg 16080ccattgaatt ttagacctgg
ctttcctggt cccagtcttg gccctggcat ggggggtggt 16140ggagggagaa
aagagttcca tggagcagat ttgggcttga tgttatctga tttatttcca
16200ggagacctgg agttctcact ttcatctgtt gaaacttggc tttcattttc
attctcttga 16260gcattctgtt ctatattatt agctacttca cagattgggg
aaagtagatc ggacagattt 16320tgctcctctc tatttccata tccagtgtaa
accacaacac aggtttctct cttaaaatca 16380attgaagcaa tggtagctgg
gtaaatgcaa ccgtcttctg accaaatggc agaacatttg 16440tccccaactt
tccactgttg taaggaagct gcagtattct tcttttggct tttattcttc
16500ttagcaggtt ttcttttagg tgtggttttt ggtttacccg aagtttcaca
aatgtcacca 16560ttctttagag catgcttaaa tgaagccaca gctttatcat
atgcttttat cagtgctgta 16620tcatcccaaa tgtcagaatc atcgctctgg
cctgtgccgc gccggaacag cacggaatcc 16680tcctgctccg ggacgccgcc
accactgccg ccgctgctca tcgccatagc aaacccgcgg 16740gtgcgcagcg
tggggccccg tcccttctta agagtgacga cttccgccgc ccggggcttc
16800tgggagcgga acagtacggt ggccgggagg accgcttgta gtaacttctc
acgctttcta 16860cgagtggtta tcgccctccc acatttgtgg cgtgtatatt
tttcatttct ctcaatcctt 16920tcatttcact gtgttatatt tcctttcctt
ttttttttgt ttgtttgttt tgagacagag 16980cctcgccctg tcgctcaggc
tggagtgcag cggcgcgatc tcggctcact gcagcctcga 17040cttcttgggc
tcaagcgatc ctcccacctc agcctcccca gtagctagga ctataggcgt
17100gcgccaccac gctcagctat tttttgtatt tagtagagac ggggtttcgg
catgttgctt 17160aggcctcgtc tcgaactcca gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt 17220agatatttat tccccctccc ccttggaaaa
gtaaatgtaa gctcctacta ggaatttaaa 17280acctgcttga tctatataaa
gacaaacaag gaaagacaaa catgggggca ggaaggaagg 17340cggcagatcc
ttaaacacta gaagatattt gatcccccaa ccttatttgt tgtttgtttt
17400gagacggagt ctcgctctgt cgtcagagtg cagtggcacc atctcggctc
attgcagcct 17460cgacctcccg agctcaagcg atcctcccgc ctcaacctcc
caagtagcta ggaccacagg 17520ggcacgccac cacacccggc tagtttctgt
atgttttgta gaggcggcgt ttggagcata 17580ttgtgtaggc tggtctcgaa
ctcctgagct caagatattc cgcccgcctc tggcatccca 17640aaatgctggg
attacaggtg tgagccacct cgcccagcct ccagtattct tttttttttt
17700tgcgacagag tattgctctg tcacccaggc tggaatgcag tggcgtgatc
tcagctcact 17760gcaacctctg cctcccaggt tcaagcaatt ctcctgcctc
agccccccga gtagctggga 17820ttacaggcgc ccaccaccac acccggctaa
tttttgtatt tttagtaaag atggggtttc 17880accatgttgg ccaggctggt
cttgaactcc tgaccttgta atccgaccgc ctcggcctcc 17940caaagtgctg
ggattacagg tgtgagccac cacaccgggc ctccagtatt ctttattaag
18000catctagggt tgctaaatgg cttatatgta catagtatat atatattttt
aactccacga 18060aaggaacttt gagctcttcc cccaaaatac ccttggcttc
tatatagtat acaagaaata 18120tctgtggagg aaggggagaa tgggatgatg
ttgaccaagt gtacaaaaat ggtaactccg 18180tagaggtaat atgtggaatg
taatcatttc acaatgtata tctaaacatc aaatggtaca 18240ccttaaatat
atacaatttt taggggtctg gtacggtggc tcatgcctat aatcccagca
18300ctttgggagg ccaaggtggg tggatcactt gaggtcagga gttcaagacc
agcctggcca 18360acatggtgaa accctgtttc tcctaaaaat acaaaaatca
gccgggtgtg gtggtgcagg 18420cctgtaatga cagctgcttg ggaggctgag
gcaggagaat ctcttgaact cgggaggcgg 18480aggttgcagt gagccaagat
cacgccactg cactccagcc tgagtgacag agtgcgactc 18540catctcaaac
aaataaatat gtacaatttt tatgtgtcaa aaaagttaaa ttgtcacaag
18600ataaaaaaaa aaattttaaa tctcatgtca ggaaagtaat gtgccaaagg
tacatctcac 18660agataaacat gaaaacctgc actccagcct gggcgacaga
gtgaggctgt gtctcagaaa 18720aaaaaaaaaa agtaaaaaaa aaagtatgtt
tttataaagc ttgcttagat ttttctgaat 18780cataaaaatt ctcacaattg
catttgatgt caaaatttaa acaaattacg tggacatatt 18840acatgatggt
taaaaaaata aatttaaaca aaatatagaa ccaggtttct ttttgttttt
18900taattttttt ctttttgaga cggagtctcg ctctgccacc cagactggag
tgcagtggct 18960cactgcaacc tctgcctccc gggttcaagt gattctcctg
tctcagcttc ccgagtacct 19020gggattacag gcgtgtgcca ccacgcccag
ctaatttttg tatttttagt agagacgggg 19080ttttgccatg ttggtcaggt
tggtctcaaa ctcctgacct tgtgatccgc ccgcctcagc 19140ctcccaaagt
gctgcgatta caggcatgag ccaccgcacc cagccatttc tttttgtttt
19200tattatttag agatataatt gatatactat agaattaatc gttttagaga
gtacaattga 19260atggtagata gagcggaaac cttaatatat tcacaaggtt
gtgcaaccat cactactatc 19320taactccaga acattttaat cacccaccaa
agaaactctg tttcctttag cagtgcgctg 19380ccatgctcag ctattttttg
ggagagaagg ggtctcccca tgttgtccac gctggtctca 19440aactcggttg
cttaagcagt cctcccactt gagccgctgt gcccaggcct gagttactat
19500atttataaaa gttatttcat atgatagaca aatcattcaa aacataatga
ggtaaactgc 19560caaaagaaac cattttacca tatttgaagg catttaatgt
aaatgttgaa tttaatttca 19620tgtactggaa tcagtctttt tgcatatgta
attttcatac caaaaatctg tcttcagttg 19680actcctggaa ctctctcatg
ataaaataaa agtttcaaat aatgtcgggg tggtggctaa 19740cacctgtaat
cccagcactg tgggagtccg aggcaggtgg atcacatgag gtcaggagtt
19800tgagaccagc ctagccaaca tggcaacact aaagatatga aagtcagcca
ggcatggtgg 19860tgcatgcctg taatctcagc tactagggag gctgaggcac
aaaaatcact tgaaactggg 19920aggtggaggt tgcaatgagc tgagatcgtg
ccactgcaca ccagcctgtg agacagagca 19980agactctgtc tcaaaaaaaa
aaaaaaaaaa aaaaaaaaaa gggccaagta tggtggctca 20040tgcctgtaat
cctagcactt tgggaggctg agtgggagag gatcatttga gcccaagtaa
20100catggtcagg ccccatctct acaaaaataa attagctggg catggtggta
tgggcttgtg 20160gtacatttat attggctcat gtccaatatg accgccat
20198920200DNAArtificial SequenceSynthetic 9gttgacattg attattgact
agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc
gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc
ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
180ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
ttggcagtac 240atcaagtgta tcatatgcca agtccgcccc ctattgacgt
caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttac
gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc
atggtgatgc ggttttggca gtacaccaat gggcgtggat 420agcggtttga
ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc
ccgttgacgc 540aaatgggcgg taggcgtgta cggtgggagg tctatataag
cagagctcgt ttagtgaacc 600gtcagatctc tagaagctgg gtaccagctg
ctagccacca tggcttccaa ggtgtacgac 660cccgagcaac gcaaacgcat
gatcactggg cctcagtggt gggctcgctg caagcaaatg 720aacgtgctgg
actccttcat caactactat gattccgaga agcacgccga gaacgccgtg
780atttttctgc atggtaacgc tgcctccagc tacctgtgga ggcacgtcgt
gcctcacatc 840gagcccgtgg ctagatgcat catccctgat ctgatcggaa
tgggtaagtc cggcaagagc 900gggaatggct catatcgcct cctggatcac
tacaagtacc tcaccgcttg gttcgagctg 960ctgaaccttc caaagaaaat
catctttgtg ggccacgact ggggggcttg tctggccttt 1020cactactcct
acgagcacca agacaagatc aaggccatcg tccatgctga gagtgtcgtg
1080gacgtgatcg agtcctggga cgagtggcct gacatcgagg aggatatcgc
cctgatcaag 1140agcgaagagg gcgagaaaat ggtgcttgag aataacttct
tcgtcgagac catgctccca 1200agcaagatca tgcggaaact ggagcctgag
gagttcgctg cctacctgga gccattcaag 1260gagaagggcg aggttagacg
gcctaccctc tcctggcctc gcgagatccc tctcgttaag 1320ggaggcaagc
ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc
1380gacgatctgc ctaagatgtt catcgagtcc gaccctgggt tcttttccaa
cgctattgtc 1440gagggagcta agaagttccc taacaccgag ttcgtgaagg
tgaagggcct ccacttcagc 1500caggaggacg ctccagatga aatgggtaag
tacatcaaga gcttcgtgga gcgcgtgctg 1560aagaacgagc agtaattcta
gagcggccgc tcgaggccgg caaggccgga tccagacatg 1620ataagataca
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
1680atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
caataaacaa 1740gttaacaaca acaattgcat tcattttatg tttcaggttc
agggggaggt gtgggaggtt 1800ttttaaagca agtaaaacct ctacaaatgt
ggtatggctg attatgatcc ggctgcctcg 1860cgcgtttcgg tgatgacggt
gaaaacctct gacacatgca gctcccggag acggtcacag 1920cttgtctgta
agcggatgcc gggagcagac aagcccgtca ggcgtcagcg ggtgttggcg
1980ggtgtcgggg cgcagccatg aggtcgactc tagaggatcg atgccccgcc
ccggacgaac 2040taaacctgac tacgacatct ctgccccttc ttcgcggggc
agtgcatgta atcccttcag 2100ttggttggta caacttgcca actgggccct
gttccacatg tgacacgggg ggggaccaaa 2160cacaaagggg ttctctgact
gtagttgaca tccttataaa tggatgtgca catttgccaa 2220cactgagtgg
ctttcatcct ggagcagact ttgcagtctg tggactgcaa cacaacattg
2280cctttatgtg taactcttgg ctgaagctct tacaccaatg ctgggggaca
tgtacctccc 2340aggggcccag gaagactacg ggaggctaca ccaacgtcaa
tcagaggggc ctgtgtagct 2400accgataagc ggaccctcaa gagggcatta
gcaatagtgt ttataaggcc cccttgttaa 2460ccctaaacgg gtagcatatg
cttcccgggt agtagtatat actatccaga ctaaccctaa 2520ttcaatagca
tatgttaccc aacgggaagc atatgctatc gaattagggt tagtaaaagg
2580gtcctaagga acagcgatat ctcccacccc atgagctgtc acggttttat
ttacatgggg 2640tcaggattcc acgagggtag tgaaccattt tagtcacaag
ggcagtggct gaagatcaag 2700gagcgggcag tgaactctcc tgaatcttcg
cctgcttctt cattctcctt cgtttagcta 2760atagaataac tgctgagttg
tgaacagtaa ggtgtatgtg aggtgctcga aaacaaggtt 2820tcaggtgacg
cccccagaat aaaatttgga cggggggttc agtggtggca ttgtgctatg
2880acaccaatat aaccctcaca aaccccttgg gcaataaata ctagtgtagg
aatgaaacat 2940tctgaatatc tttaacaata gaaatccatg gggtggggac
aagccgtaaa gactggatgt 3000ccatctcaca cgaatttatg gctatgggca
acacataatc ctagtgcaat atgatactgg 3060ggttattaag atgtgtccca
ggcagggacc aagacaggtg aaccatgttg ttacactcta 3120tttgtaacaa
ggggaaagag agtggacgcc gacagcagcg gactccactg gttgtctcta
3180acacccccga aaattaaacg gggctccacg ccaatggggc ccataaacaa
agacaagtgg 3240ccactctttt ttttgaaatt gtggagtggg ggcacgcgtc
agcccccaca cgccgccctg 3300cggttttgga ctgtaaaata agggtgtaat
aacttggctg attgtaaccc cgctaaccac 3360tgcggtcaaa ccacttgccc
acaaaaccac taatggcacc ccggggaata cctgcataag 3420taggtgggcg
ggccaagata ggggcgcgat tgctgcgatc tggaggacaa attacacaca
3480cttgcgcctg agcgccaagc acagggttgt tggtcctcat attcacgagg
tcgctgagag 3540cacggtgggc taatgttgcc atgggtagca tatactaccc
aaatatctgg atagcatatg 3600ctatcctaat ctatatctgg gtagcatagg
ctatcctaat ctatatctgg gtagcatatg 3660ctatcctaat ctatatctgg
gtagtatatg ctatcctaat ttatatctgg gtagcatagg 3720ctatcctaat
ctatatctgg gtagcatatg ctatcctaat ctatatctgg gtagtatatg
3780ctatcctaat ctgtatccgg gtagcatatg ctatcctaat agagattagg
gtagtatatg 3840ctatcctaat ttatatctgg gtagcatata ctacccaaat
atctggatag catatgctat 3900cctaatctat atctgggtag catatgctat
cctaatctat atctgggtag cataggctat 3960cctaatctat atctgggtag
catatgctat cctaatctat atctgggtag tatatgctat 4020cctaatttat
atctgggtag cataggctat cctaatctat atctgggtag catatgctat
4080cctaatctat atctgggtag tatatgctat cctaatctgt atccgggtag
catatgctat 4140cctcatgcat atacagtcag catatgatac ccagtagtag
agtgggagtg ctatcctttg 4200catatgccgc cacctcccaa gggggcgtga
attttcgctg cttgtccttt tcctgctggt 4260tgctcccatt cttaggtgaa
tttaaggagg ccaggctaaa gccgtcgcat gtctgattgc 4320tcaccaggta
aatgtcgcta atgttttcca acgcgagaag gtgttgagcg cggagctgag
4380tgacgtgaca acatgggtat gcccaattgc cccatgttgg gaggacgaaa
atggtgacaa 4440gacagatggc cagaaataca ccaacagcac gcatgatgtc
tactggggat ttattcttta 4500gtgcggggga atacacggct tttaatacga
ttgagggcgt ctcctaacaa gttacatcac 4560tcctgccctt cctcaccctc
atctccatca cctccttcat ctccgtcatc tccgtcatca 4620ccctccgcgg
cagccccttc caccataggt ggaaaccagg gaggcaaatc tactccatcg
4680tcaaagctgc acacagtcac cctgatattg caggtaggag cgggctttgt
cataacaagg 4740tccttaatcg catccttcaa aacctcagca aatatatgag
tttgtaaaaa gaccatgaaa 4800taacagacaa tggactccct tagcgggcca
ggttgtgggc cgggtccagg ggccattcca 4860aaggggagac gactcaatgg
tgtaagacga cattgtggaa tagcaagggc agttcctcgc 4920cttaggttgt
aaagggaggt cttactacct ccatatacga acacaccggc gacccaagtt
4980ccttcgtcgg tagtcctttc tacgtgactc ctagccagga gagctcttaa
accttctgca 5040atgttctcaa atttcgggtt ggaacctcct tgaccacgat
gcttttccaa accaccctcc 5100ttttttgcgc cctgcctcca tcaccctgac
cccggggtcc agtgcttggg ccttctcctg 5160ggtcatctgc ggggccctgc
tctatcgctc ccgggggcac gtcaggctca ccatctgggc 5220caccttcttg
gtggtattca aaataatcgg cttcccctac agggtggaaa aatggccttc
5280tacctggagg gggcctgcgc ggtggagacc cggatgatga tgactgacta
ctgggactcc 5340tgggcctctt ttctccacgt ccacgacctc tccccctggc
tctttcacga cttccccccc 5400tggctctttc acgtcctcta ccccggcggc
ctccactacc tcctcgaccc cggcctccac 5460tacctcctcg accccggcct
ccactgcctc ctcgaccccg gcctccacct cctgctcctg 5520cccctcctgc
tcctgcccct cctcctgctc ctgcccctcc tgcccctcct gctcctgccc
5580ctcctgcccc tcctgctcct gcccctcctg cccctcctgc tcctgcccct
cctgcccctc 5640ctcctgctcc tgcccctcct gcccctcctc ctgctcctgc
ccctcctgcc cctcctgctc 5700ctgcccctcc tgcccctcct gctcctgccc
ctcctgcccc tcctgctcct gcccctcctg 5760ctcctgcccc tcctgctcct
gcccctcctg ctcctgcccc tcctgcccct cctgcccctc 5820ctcctgctcc
tgcccctcct gctcctgccc ctcctgcccc tcctgcccct cctgctcctg
5880cccctcctcc tgctcctgcc cctcctgccc ctcctgcccc tcctcctgct
cctgcccctc 5940ctgcccctcc tcctgctcct gcccctcctc ctgctcctgc
ccctcctgcc cctcctgccc 6000ctcctcctgc tcctgcccct cctgcccctc
ctcctgctcc tgcccctcct cctgctcctg 6060cccctcctgc ccctcctgcc
cctcctcctg ctcctgcccc tcctcctgct cctgcccctc 6120ctgcccctcc
tgcccctcct gcccctcctc ctgctcctgc ccctcctcct gctcctgccc
6180ctcctgctcc tgcccctccc gctcctgctc ctgctcctgt tccaccgtgg
gtccctttgc 6240agccaatgca acttggacgt ttttggggtc tccggacacc
atctctatgt cttggccctg 6300atcctgagcc gcccggggct cctggtcttc
cgcctcctcg tcctcgtcct cttccccgtc 6360ctcgtccatg gttatcaccc
cctcttcttt gaggtccact gccgccggag ccttctggtc 6420cagatgtgtc
tcccttctct cctaggccat ttccaggtcc tgtacctggc ccctcgtcag
6480acatgattca cactaaaaga gatcaataga catctttatt agacgacgct
cagtgaatac 6540agggagtgca gactcctgcc ccctccaaca gcccccccac
cctcatcccc ttcatggtcg 6600ctgtcagaca gatccaggtc tgaaaattcc
ccatcctccg aaccatcctc gtcctcatca 6660ccaattactc gcagcccgga
aaactcccgc tgaacatcct caagatttgc gtcctgagcc 6720tcaagccagg
cctcaaattc ctcgtccccc tttttgctgg acggtaggga tggggattct
6780cgggacccct cctcttcctc ttcaaggtca ccagacagag atgctactgg
ggcaacggaa 6840gaaaagctgg gtgcggcctg tgaggatcag cttatcgatg
ataagctgtc aaacatgaga 6900attcttgaag acgaaagggc ctcgtgatac
gcctattttt ataggttaat gtcatgataa 6960taatggtttc ttagacgtca
ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 7020gtttattttt
ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
7080tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
gtcgccctta 7140ttcccttttt tgcggcattt tgccttcctg tttttgctca
cccagaaacg ctggtgaaag 7200taaaagatgc tgaagatcag ttgggtgcac
gagtgggtta catcgaactg gatctcaaca 7260gcggtaagat ccttgagagt
tttcgccccg aagaacgttt tccaatgatg agcactttta 7320aagttctgct
atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
7380gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
gaaaagcatc 7440ttacggatgg catgacagta agagaattat gcagtgctgc
cataaccatg agtgataaca 7500ctgcggccaa cttacttctg acaacgatcg
gaggaccgaa ggagctaacc gcttttttgc 7560acaacatggg ggatcatgta
actcgccttg atcgttggga accggagctg aatgaagcca 7620taccaaacga
cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
7680tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
tggatggagg 7740cggataaagt tgcaggacca cttctgcgct cggcccttcc
ggctggctgg tttattgctg 7800ataaatctgg agccggtgag cgtgggtctc
gcggtatcat tgcagcactg gggccagatg 7860gtaagccctc ccgtatcgta
gttatctaca cgacggggag tcaggcaact atggatgaac 7920gaaatagaca
gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
7980aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
aaaaggatct 8040aggtgaagat cctttttgat aatctcatga ccaaaatccc
ttaacgtgag ttttcgttcc 8100actgagcgtc agaccccgta gaaaagatca
aaggatcttc ttgagatcct ttttttctgc 8160gcgtaatctg ctgcttgcaa
acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 8220atcaagagct
accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
8280atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
gtagcaccgc 8340ctacatacct cgctctgcta atcctgttac cagtggctgc
tgccagtggc gataagtcgt 8400gtcttaccgg gttggactca agacgatagt
taccggataa ggcgcagcgg tcgggctgaa 8460cggggggttc gtgcacacag
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 8520tacagcgtga
gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
8580cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
ggaaacgcct 8640ggtatcttta tagtcctgtc gggtttcgcc acctctgact
tgagcgtcga tttttgtgat 8700gctcgtcagg ggggcggagc ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc 8760tggccttttg ctggccttga
agctgtccct gatggtcgtc atctacctgc ctggacagca 8820tggcctgcaa
cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
8880ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg
gccccgagat 8940gcgccgcgtg cggctgctgg agatggcgga cgcgatggat
atgttctgcc aagggttggt 9000ttgcgcattc acagttctcc gcaagaattg
attggctcca attcttggag tggtgaatcc 9060gttagcgagg tgccgccctg
cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg 9120tccccggaag
aaatatattt gcatgtcttt agttctatga tgacacaaac cccgcccagc
9180gtcttgtcat tggcgaattc
gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc 9240acttcgcata
ttaaggtgac gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg
9300cttaacagcg tcaacagcgt gccgcagatc ccggggggca atgagatatg
aaaaagcctg 9360aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
gttcgacagc gtctccgacc 9420tgatgcagct ctcggagggc gaagaatctc
gtgctttcag cttcgatgta ggagggcgtg 9480gatatgtcct gcgggtaaat
agctgcgccg atggtttcta caaagatcgt tatgtttatc 9540ggcactttgc
atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
9600agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
gacctgcctg 9660aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
catggatgcg atcgctgcgg 9720ccgatcttag ccagacgagc gggttcggcc
cattcggacc gcaaggaatc ggtcaataca 9780ctacatggcg tgatttcata
tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 9840tgatggacga
caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
9900ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
aacaatgtcc 9960tgacggacaa tggccgcata acagcggtca ttgactggag
cgaggcgatg ttcggggatt 10020cccaatacga ggtcgccaac atcttcttct
ggaggccgtg gttggcttgt atggagcagc 10080agacgcgcta cttcgagcgg
aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 10140atatgctccg
cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
10200atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
gggactgtcg 10260ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
cgatggctgt gtagaagtac 10320tcgccgatag tggaaaccga cgccccagca
ctcgtccgga tcgggagatg ggggaggcta 10380actgaaacac ggaaggagac
aataccggaa ggaacccgcg ctatgacggc aataaaaaga 10440cagaataaaa
cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
10500ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
gcgtttcttc 10560cttttcccca ccccaccccc caagttcggg tgaaggccca
gggctcgcag ccaacgtcgg 10620ggcggcaggc cctgccatag ccactggccc
cgtgggttag ggacggggtc ccccatgggg 10680aatggtttat ggttcgtggg
ggttattatt ttgggcgttg cgtggggtca ggtccacgac 10740tggactgagc
agacagaccc atggtttttg gatggcctgg gcatggaccg catgtactgg
10800cgcgacacga acaccgggcg tctgtggctg ccaaacaccc ccgaccccca
aaaaccaccg 10860cgcggatttc tggcgtgcca agctagtcga ccaattctca
tgtttgacag cttatcatcg 10920cagatccggg caacgttgtt gccattgctg
caggcgcaga actggtaggt atggaagatc 10980tatacattga atcaatattg
gcaattagcc atattagtca ttggttatat agcataaatc 11040aatattggct
attggccatt gcatacgttg tatctatatc ataatatgta cctaaccaag
11100ttcctctttc agaggttatt tcaggccatg gtgctgcgca gatccgcgta
tgcggtgtga 11160aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
cgaaattgta aacgttaata 11220ttttgttaaa attcgcgtta aatatttgtt
aaatcagctc attttttaac caataggccg 11280aaatcggcaa aatcccttat
aaatcaaaag aatagaccga gatagggttg agtgttgttc 11340cagtttggaa
caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
11400ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt
tttttgcggt 11460cgaggtgccg taaagctcta aatcggaacc ctaaagggag
cccccgattt agagcttgac 11520ggggaaagcc ggcgaacgtg gcgagaaagg
aagggaagaa agcgaaagga gcgggcgcta 11580gggcgctggc aagtgtagcg
gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 11640cgccgctaca
gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
11700ggtgcgggcc tcttcgctat tacgccagcc cggatcgatc cttatcggat
tttaccacat 11760ttgtagaggt tttacttgct ttaaaaaacc tcccacatct
ccccctgaac ctgaaacata 11820aaatgaatgc aattgttgtt gttaacttgt
ttattgcagc ttataatggt tacaaataaa 11880gcaatagcat cacaaatttc
acaaataaag catttttttc actgcattct agttgtggtt 11940tgtccaaact
catcaatgta tcttatcatg tctgctcgaa gcattaaccc tcactaaagg
12000gaagcggccg cttacatttt acttacaatt tggactttcc gcccttcttg
gcctttatga 12060ggatctctct gatttttctt gcgtcgagtt ttccggtaag
acctttcggt acttcgtcca 12120caaacacaac tcctccgcgc aactttttcg
cggttgttac ttgactggcg acgtaatcca 12180cgatctcttt ttccgtcatc
gtctttccgt gctccaaaac aacaacggcg gcgggaagtt 12240caccggcgtc
atcgtcggga agacctgcca cgcccgcgtc gaagatgttg gggtgttgta
12300acaatatcga ttccaattca gcgggggcca cctgatatcc tttgtattta
attaaagact 12360tcaagcggtc aactatgaag aagtgttcgt cttcgtccca
gtaagctatg tctccagaat 12420gtagccatcc atccttgtca atcaaggcgt
tggtcgcttc cggattgttt acataaccgg 12480acataatcat aggtcctctg
acacataatt cgcctctctg attaacgccc agcgttttcc 12540cggtatccag
atccacaacc ttcgcttcaa aaaatggaac aactttaccg accgcgcccg
12600gtttatcatc cccctcgggt gtaatcagaa tagctgatgt agtctcagtg
agcccatatc 12660cttgtcgtat ccctggaaga tggaagcgtt ttgcaaccgc
ttccccgact tctttcgaaa 12720gaggtgcgcc cccagaagca atttcgtgta
aattagataa atcgtatttg tcaatcagag 12780tgcttttggc gaagaatgaa
aatagggttg gtactagcaa cgcactttga attttgtaat 12840cctgaaggga
tcgtaaaaac agctcttctt caaatctata cattaagacg actcgaaatc
12900cacatatcaa atatccgagt gtagtaaaca ttccaaaacc gtgatggaat
ggaacaacac 12960ttaaaatcgc agtatccgga atgatttgat tgccaaaaat
aggatctctg gcatgcgaga 13020atctgacgca ggcagttcta tgcggaaggg
ccacaccctt aggtaaccca gtagatccag 13080aggaattcat tatcagtgca
attgttttgt cacgatcaaa ggactctggt acaaaatcgt 13140attcattaaa
accgggaggt agatgagatg tgacgaacgt gtacatcgac tgaaatccct
13200ggtaatccgt tttagaatcc atgataataa ttttctggat tattggtaat
tttttttgca 13260cgttcaaaat tttttgcaac ccctttttgg aaacaaacac
tacggtaggc tgcgaaatgt 13320tcatactgtt gagcaattca cgttcattat
aaatgtcgtt cgcgggcgca actgcaactc 13380cgataaataa cgcgcccaac
accggcataa agaattgaag agagttttca ctgcatacga 13440cgattctgtg
atttgtattc agcccatatc gtttcatagc ttctgccaac cgaacggaca
13500tttcgaagta ttccgcgtac gtgatgttca cctcgatatg tgcatctgta
aaagcaattg 13560ttccaggaac cagggcgtat ctcttcatag ccttatgcag
ttgctctcca gcggttccat 13620cctctagagg atagaatggc gccgggcctt
tctttatgtt tttggcgtct tccagctgct 13680ctatgccagc atttcctgca
aatgagaaat tagaaccaga ggcttgacga attccagtta 13740aaccatgtcc
tctgtggaca ccagttaaac ttgactagag cacttcatat gtcagagtgt
13800acagtgcagt atgcctaggt tatcccatat cacaataaaa aaaagtctgc
tggtctgcct 13860actagtgata taaaatggca tcatatccta aagctcttta
ttgtgaaagt atgtttcttc 13920cacacaacca accagttaag tatgagaatt
ctagtaggga tgtagattaa ccttttatct 13980aatagttttg gcatcaaaat
tctttaatat tgattgtttt acattaacct ttcaactttc 14040taacatctga
actttttaaa tgttcaaaaa catttgtttt ccacaaacca taaagtttta
14100caaaagtaag attcactttc ataatgctgg cagacttact ccttagattt
aaggaatgtg 14160agcaccttcc ttctttttga ttttgtctaa aaccctgtaa
ggaaaataaa ggaagttaaa 14220aaaaatagct atatagatat agatagctat
atatagatag ctttatatgg atgttaaaaa 14280gcattttgtt tcacaagaca
ttttacttat tttattcaac aaaatatgat cagaaattaa 14340gttgatagtc
ttttaatgta ctttaaaagt tatcccaaag aaaacaatta ttaggctgca
14400gttaaggttt tcttgcagtg gctcatgcct acaatcccac aactttggga
ggcggaggta 14460gggggatcac ttgagacctg gagcttgaca ccaccctggg
caacataatg agaccctgtc 14520tctacaaaaa atttaaaaat taggccggcg
tggtggctca tgctaggcac agtggctcac 14580gcctgtaatc ccagcacttt
gggaggccga gacagttgga tcacctgagc tcaggagttc 14640gagaacagcc
tggccaacat ggcaaaaccc cgtttctact gaaagtacaa aaaattagcc
14700aggcatggtg gtggggacct ctaatcccag ctacttggga ggttgaggca
ggagaatcac 14760ttgaacccag gaggcggagg ctgcagtgag ctgagattta
caccactgca ctccagcctg 14820ggtgacagag caagactctg tctcaaaaaa
aaataaataa ataaaaataa aaattagcca 14880ggtgcagtgg cattatcact
gtagtcccag ctactcggga aactgaggtg agaggactgc 14940ttgagccctg
gaggtcaagg ctgcagtgag ctgtgaatgt gcccttgcac tccagcctga
15000gcaatagagt gagacctggt ctctaaaaaa taaaatttgg gaggcggagc
ttgcagtgag 15060ccgagactgc gccactgtac tccagcctgg gtgacagagc
gagactccgt ctcaaaaaaa 15120aaaaaaaaat aaaataaaat aaaataattt
taaatgttct gactaaaata caatagaaca 15180tgtccgtagg agactaacgt
ataaagtgac aagtttgaag ccatactccc caaggttcaa 15240tgtggtacac
attaccccag atctttgtgc attaaaaaaa tttcatttct cttggaaggc
15300cgaggcgggt ggatcacggg gtcaggagat tgagaccatc ctggctaaca
cagtgaaacc 15360ctgtctttac aaaaaaatac aaaaaattag acaggcgtgg
tggcaggcaa ctgtagtccc 15420agctacctct gaggctgagg caggagaatg
gcgtgaatcc aggaggcaga gcttgctgtg 15480agccaagatc acgccattgc
actccagcct gggcaacaga gcaagactcc gtctcaaaaa 15540aaaaaaaaaa
aaaaaagaat ttcatttttc atttatgaaa aattatccca tcttttccat
15600tccctacaat caatttcaaa tcagagatta aaacattatt tagaaaaagt
ataatttcaa 15660ttcaaaagtg tataatcaaa ataatctaac aatagcatga
aagcttttta aaattaacta 15720aaattatact tagggacaat gcaagagtaa
tttaagcctc agacagttgt atttttttat 15780ttttattttt tagtaatata
aagagagaag caagtagtat tttataaatt tacaaaacaa 15840agtcacataa
ctacaaaaaa attgtcagga aaagatgctg agtgattact taccatataa
15900tagccagtat gatagccact catgtaccat gaaattaaca tacttcccaa
agcatcagca 15960tcatcaagag aatctggaca tatgggaggt ggtgggggaa
ttatctcgag tggtccagaa 16020ggaaatggag gcagccagca tgatagtaag
tggggtggtg gtggtggcgg tggcggtggt 16080gggccattga attttagacc
tggctttcct ggtcccagtc ttggccctgg catggggggt 16140ggtggaggga
gaaaagagtt ccatggagca gatttgggct tgatgttatc tgatttattt
16200ccaggagacc tggagttctc actttcatct gttgaaactt ggctttcatt
ttcattctct 16260tgagcattct gttctatatt attagctact tcacagattg
gggaaagtag atcggacaga 16320ttttgctcct ctctatttcc atatccagtg
taaaccacaa cacaggtttc tctcttaaaa 16380tcaattgaag caatggtagc
tgggtaaatg caaccgtctt ctgaccaaat ggcagaacat 16440ttgtccccaa
ctttccactg ttgtaaggaa gctgcagtat tcttcttttg gcttttattc
16500ttcttagcag gttttctttt aggtgtggtt tttggtttac ccgaagtttc
acaaatgtca 16560ccattcttta gagcatgctt aaatgaagcc acagctttat
catatgcttt tatcagtgct 16620gtatcatccc aaatgtcaga atcatcgctc
tggcctgtgc cgcgccggaa cagcacggaa 16680tcctcctgct ccgggacgcc
gccaccactg ccgccgctgc tcatcgccat agcaaacccg 16740cgggtgcgca
gcgtggggcc ccgtcccttc ttaagagtga cgacttccgc cgcccggggc
16800ttctgggagc ggaacagtac ggtggccggg aggaccgctt gtagtaactt
ctcacgcttt 16860ctacgagtgg ttatcgccct cccacatttg tggcgtgtat
atttttcatt tctctcaatc 16920ctttcatttc actgtgttat atttcctttc
cttttttttt tgtttgtttg ttttgagaca 16980gagcctcgcc ctgtcgctca
ggctggagtg cagcggcgcg atctcggctc actgcagcct 17040cgacttcttg
ggctcaagcg atcctcccac ctcagcctcc ccagtagcta ggactatagg
17100cgtgcgccac caagctcagc tattttttgt atttagtaga gacggggttt
cggcatgttg 17160cttaggcctc gtctcgaact ccagtgtgtg tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg 17220tgtgtgtgta gatatttatt ccccctcccc
cttggaaaag taaatgtaag ctcctactag 17280gaatttaaaa cctgcttgat
ctatataaag acaaacaagg aaagacaaac atgggggcag 17340gaaggaaggc
agatccttaa acactagaag atatttgatc ccccaacctt atttgttgtt
17400tgttttgaga cggagtctcg ctctgtcgtc agagtgcagt ggcaccatct
cggctcattg 17460cagcctcgac ctcccgagct caagcgatcc tcccgcctca
acctcccaag tagctaggac 17520cacaggggca cgccaccaca cccggctagt
ttctgtatgt tttgtagagg cggcgtttgg 17580agcatattgt gtaggctggt
ctcgaactcc tgagctcaag atattccgcc cgcctctggc 17640atcccaaaat
gctgggatta caggtgtgag ccacctcgcc cagcctccag tattcttttt
17700tttttttgcg acagagtatt gctctgtcac ccaggctgga atgcagtggc
gtgatctcag 17760ctcactgcaa cctctgcctc ccaggttcaa gcaattctgc
ctcagccccc cgagtagctg 17820ggattacagg cgcccaccac cacacccggc
taatttttgt atttttagta aagatggggt 17880ttcaccatgt tggccaggct
ggtcttgaac tcctgacctc gtaatccgac cgcctcggcc 17940tcccaaagtg
ctgggattac aggtgtgagc caccacaccg ggcctccagt attctttatt
18000aagcatctag ggttgctaaa tggcttatat gtacatagta tatatatatt
tttaactcca 18060cgaaaggaac tttgagctct tcccccaaaa tacccttggc
ttctatatag tatacaagaa 18120atatctgtgg aggaagggga gaatgggatg
atgttgacca agtgtacaaa aatggtaact 18180ctgtagaggt aatatgtgga
atgtaatcat ttcacaatgt atatctaaac atcaaatggt 18240acaccttaaa
tatatacaat ttttaggggt ctggtacggt ggctcatgcc tataatccca
18300gcactttggg aggccaaggt gggtggatca cttgaggtca ggacttcaag
accagcctgg 18360ccaacatggt gaaaccctgt ttctcctaaa aatacaaaaa
tcagccgggt gtggtggtgc 18420aggcctgtaa tgacagctgc ttgggaggct
gagccaggag aatcacttga actcgggagg 18480cggaggttgc agtgagccaa
gatcacgcca ctgcactcca gcctgagtga cagagtgcga 18540ctccatctca
aacaaataaa tatgtacaat ttttatgtgt caaaaaagtt aaattgtcac
18600aagataaaaa aaaaaattta aatctcatgt caggaaagta atgtgccaaa
ggtacatctc 18660acagataaac atgaaaacct gcactccagc ctgggcgaca
gagtgaggct gtgtctcaga 18720aaaaaaaaaa aaagtaaaaa aaaaagtatg
tttttataaa gcttgcttag atttttctga 18780atcataaaaa ttctcacaat
tgcatttgat gtcaaaattt aaacaaatta cctggacata 18840ttacatgatg
gttaaaaaaa taaatttaaa caaaatatag aaccaggttt ctttttgttt
18900tttaattttt ttctttttga gacggagtct cgctctgcca cccagactgg
agtgcagtgg 18960ctcactgcaa cctctgcctc ccgggttcaa gtgattctcc
tgtctcagct tcccgagtac 19020ctaggattac aggcgtgtgc caccacaccc
agctaatttt tgtattttta gtagagactg 19080ggttttgcca tgttggtcag
gttggtctca aactcctgac cttgtgatcc gcccgcctca 19140gcctcccaaa
gtgctgcgat tacaggcatg agccaccgca cccagccatt tctttttgtt
19200tttattattt agagatataa ttgatatact atagaattaa tcgttttaga
gagtacaatt 19260gaatggtaga tagagcggaa accttaatat attcacaagg
ttgtgcaacc atcactacta 19320tctaactcca gaacatttta atcacccacc
aaagaaactc tgtttccttt agcagtgcgc 19380tgccatgctc agctattttt
tgggagagaa ggggtctccc catgttgtcc acgctggtct 19440caaactcggt
tgcttaagca gtcctcccac ttgagccgct gtgcccaggc ctgagttact
19500atatttataa aagttatttc atatgataga caaatcattc aaaacataat
gaggtaaact 19560gccaaaagaa accattttac catatttgaa ggcatttaat
gtaaatgttg aatttaattt 19620catgtactgg aatcagtctt tttgcatatg
taattttcat accaaaaatc tctcttcagt 19680tgactcctgg aactctctca
tgataaaata aaagtttcaa ataatgtcgg ggtggtggct 19740aacacctgta
atcccagcac tgtgggagtc cgaggcaggt ggatcacatg aggtcaggag
19800tttgagacca gcctagccaa catggcaaca ctaaagatat gaaagtcagc
caggcatggt 19860ggtgcatgcc tgtaatctca gctactaggg aggctgaggc
acaaaaatca cttgaaactg 19920ggaggtggag gttgcaatga gctgagatcg
tgccactgca caccagcctg tgagacagag 19980caagactctg tctcaaaaaa
aaaaaaaaaa aaaaaaaaaa aagggccaag tatggtggct 20040catgcctgta
atcctagcac tttgggaggc tgagtgggag aggatcattt gagcccaagt
20100aacatggtca ggccccatct ctacaaaaat aaattagctg ggcatggtgg
tatgggcttg 20160tggtacattt atattggctc atgtccaata tgaccgccat
20200
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