(en)The invention relates to a compound of Formula I:

1.ApplicationNumber: US-201313887466-A
1.PublishNumber: US-8741905-B2
2.Date Publish: 20140603
3.Inventor: WAGNER BRIDGET
DUVALL JEREMY
CHOU DANNY HUNG-CHIEH

4.Inventor Harmonized: WAGNER BRIDGET(US)
DUVALL JEREMY(US)
CHOU DANNY HUNG-CHIEH(US)

5.Country: US
6.Claims:
(en)The invention relates to a compound of Formula I:

7.Description:
(en)RELATED APPLICATIONS
This application is a continuation of International Application No. PCT/US2011/059405, which designated the United States and was filed on Nov. 4, 2011, published in English, which claims the benefit of U.S. Provisional Application No. 61/410,558, filed on Nov. 5, 2010. The entire teachings of the above applications are incorporated herein by reference.


GOVERNMENT SUPPORT
The invention was supported, in whole or in part, by a grant DP2-DK083048 from NIH-NIDDK. The Government has certain rights in the invention.


FIELD OF THE INVENTION
This invention relates to the treatment autoimmune diseases including type-1 diabetes, lupus and rheumatoid arthritis.
BACKGROUND OF THE INVENTION
Type-1 diabetes is caused by the autoimmune destruction of insulin producing β-cells in the pancreas. β-cell apoptosis involves a set of signaling cascades initiated by interleukin-1β (IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). (Chou et al., ACS Chem. Biol. 2010 Aug. 20; 5(8):729-34). IL-1 β and TNF-α induce NFκB expression, and downstream activation of gene expression is thought to occur through nitric oxide (NO) signaling, which both increases endoplasmic reticulum stress-response pathways and decreases β cell-specific functions. NO is a highly reactive molecule that inhibits the electron-transport chain, leading to decreases in glucose oxidation rates, ATP generation, and insulin production; cellular nitrite is more stable and serves as a surrogate marker for NO. NFκB activation and IFN-α-induced STAT-1 signaling work together to effect β-cell apoptosis, mainly involving the intrinsic apoptotic pathway in both rodents and humans. The downstream effector of this cascade, caspase-3, results in apoptosis and the loss of the ability to secrete insulin in response to glucose stimulation. Cnop et al., Diabetes 54, (Suppl 2), S97-107, 2005; Formoni et al., Diabetologia 51, 298-308, 2008; Soldevila et al., J. Autoimmun. 4, 291-306, 1991; Darville et al., Diabetologia 41, 1101-1108, 1998. Kharroubi et al., Endocrinology 145, 5087-5096, 2004; Mandrup-Poulsen et al., Diabetologia 39, 1005-1029, 1996.
For decades, the standard of care for this disease has been insulin therapy via intramuscular injection. Current approaches to develop new treatments have prioritized islet transplantation) and directed stem-cell differentiation, while many technological advances have focused on glucose detection and insulin delivery. Recent development of techniques to treat diabetic mice with partially differentiated stem cells indicates that procedures resulting in an increase in beta-cell mass can ameliorate type-1 diabetes. While cell-based treatments show promise, a chemical intervention capable of restoring glycemic control in type-1 diabetes would have enormous impact clinically, by enabling an in vivo pancreatic effect while avoiding the need for immunosuppression.
SUMMARY OF THE INVENTION
The invention relates to compounds of Formula I or a prodrug or metabolite thereof:

wherein,

Each X 1 and X 3 is independently —O— or —S—;

Each X 4 , X 5 and X 6 is independently —CH or —N—;

Each X 2 , Y 1 and Y 2 is independently —NR 10 , —S— or —O—;

Cy 1 is an optionally substituted aryl group;

Cy 2 is an optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl or optionally substituted aryl containing one, two or three rings;

R 1 , is hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;

Each R 2 , R 3 , R 4 and R 5 is independently selected from absent, hydrogen, halogen, —OR 10 , —SR 10 , —NR 10 R 11 , —C(O)R 10 , —C(O)OR 10 , —C(O)NR 10 R 11 , —N(R 10 )C(O)R 11 , —CF 3 , —CN, —NO 2 , —N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl;

G 1 is —N(R 10 )C(O)—, —N(R 10 )C(S)—, —N(R 10 )S(O) 2 —, —N(R 10 )S(O) 2 —[C(R 10 )(R 11 )] W —, —N(R 10 )C(O)—[C(R 10 )(R 11 )] W —, —N(R 10 )C(S)—[C(R 10 )(R 11 )] W —, —N(R 10 )C(O)N(R 11 )—, —N(R 10 )C(S)N(R 11 )—, —C(O)O— or —C(O)O—[C(R 10 )(R 11 )] W —;

Each n and m is independently selected from 0, 1, 2 or 3;

Each v and w is independently selected from 0, 1, 2, 3, 4, 5 or 6;

Each R 10 , R 11 , R 12 , R 13 and R 14 is independently absent, hydrogen, halogen, —OH, —SH, —NH 2 , —CF 3 , —CN, —NO 2 , —N 3 , —C(O)OH, —C(O)NH 2 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of R 10 , R 11 , R 12 , R 13 and R 14 together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring.

The invention further relates to the treatment of autoimmune diseases by the administration of a compound of Formula I. Compounds of Formula I can be useful for the treatment of autoimmune diseases including multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, osteoarthritis, psoriasis, ulcerative colitis, type-1 diabetes, pancreatitis, asthma, idiopathic thrombocytopenia purpura, uveitis, Guillain-Barre syndrome or myasthenia gravis.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 : Cellular ATP Levels after incubation with Compound-1A.
FIG. 2 : Caspase activation after incubation with Compound-1A.
FIG. 3 : Glucose-stimulated insulin secretion with Compound-21A.
FIG. 4 : The effect of Compound-21A (10 μM) on beta cell apoptosis indicated by relative levels of ATP in the presence of common modes of beta-cell apoptosis: (A) ER stress induced by thapsigargin, (B) ER stress induced by tunicamycin, and (C) glucose toxicity. All three graphs show a DMSO control (♦) and Compound-21A (▪).
FIG. 5 : Gene-set enrichment analysis after 6-hr treatment with Compound-21A: (A) Enrichment plot showing SANA response to interferon-γ, (B) Enrichment plot showing Browne response to interferon-γ.
FIG. 6 : Schematic showing role of interferon-γ in STAT1-dependent signaling pathway.
FIG. 7 : Compound-21A inhibits cytokine-induced STAT1 reporter gene activity.
FIG. 8 : Suppression of STAT1 phosphorylation by Compound-21A.
FIG. 9 : Inhibition of nuclear STAT1 translocation by Compound-21A.
FIG. 10 : The effect of putative JAK2 inhibitors, SD-1029 and Z3, on cytokine-induced beta cell apoptosis.
FIG. 11 : Schematic of candidate protein binder by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).
FIG. 12 : Candidate proteins identified by SILAC and mass spectrometry analysis.
FIG. 13 : Usp9x isolated by low-throughput Compound-21A pull-down and Western blot.
FIG. 14 : Usp9x purification and thermal shift assay and comparison of Compound-21A with non-specific debiquitinase inhibitor WP1130, suggests binding.
FIG. 15 : Compound-21 enhances effect of ABT-737 on DLD-1 cell death.
FIG. 16 : STAT1 levels and phosphorylation after Usp9x knock-down.



DETAILED DESCRIPTION OF THE INVENTION

wherein,

Each X 1 and X 3 is independently —O— or —S—;

Each X 4 , X 5 and X 6 is independently —CH or —N—;

Each X 2 , Y 1 and Y 2 is independently —NR 10 , —S— or —O—;

Cy 1 is an optionally substituted aryl group;

Cy 2 is an optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl or optionally substituted aryl containing one, two or three rings;

R 1 is hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;

Each R 2 , R 3 , R 4 and R 5 is independently selected from absent, hydrogen, halogen, —OR 10 , —SR 10 , —NR 10 R 11 , —C(O)R 10 , —C(O)OR 10 , —C(O)NR 10 R 11 , —N(R 10 )C(O)R 11 , —CF 3 , —CN, —NO 2 , —N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl;

G 1 is —N(R 10 )C(O)—, —N(R 10 )C(S)—, —N(R 10 )S(O) 2 —, —N(R 10 )S(O) 2 —[C(R 10 )(R 11 )] W —, —N(R 10 )C(O)—[C(R 10 )(R 11 )] W —, —N(R 10 )C(S)—[C(R 10 )(R 11 )] W —, —N(R 10 )C(O)N(R 11 )—, —N(R 10 )C(S)N(R 11 )—, —C(O)O— or —C(O)O—[C(R 10 )(R 11 )] W —; Each n and m is independently selected from 0, 1, 2 or 3;

Each v and w is independently selected from 0, 1, 2, 3, 4, 5 or 6;

Each R 10 , R 11 , R 12 , R 13 and R 14 is independently absent, hydrogen, halogen, —OH, —SH, —NH 2 , —CF 3 , —CN, —NO 2 , —N 3 , —C(O)OH, —C(O)NH 2 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of R 10 , R 11 , R 12 , R 13 and R 14 together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring.

In a preferred embodiment Cy 1 is selected from:

wherein each R 100 , R 101 , R 102 and R 103 is independently absent, hydrogen, halogen, —OR 10 , —SR 10 , —NR 10 R 11 , —C(O)R 10 , —C(O)OR 10 , —C(O)NR 10 R 11 , —N(R 10 )C(O)R 11 , —CF 3 , —CN, —NO 2 , —N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl.

In a preferred embodiment Cy 2 is selected from;









In a preferred embodiment X 1 is —O—.
In a preferred embodiment X 2 is —O—.
In a preferred embodiment X 3 is —O—.
In a preferred embodiment X 4 is —CH.
In a preferred embodiment X 5 is —CH.
In a preferred embodiment X 6 is —CH.
In a preferred embodiment n is 1, and R 5 is —CH 3 .
In a preferred embodiment m is 0.
In a preferred embodiment Y 1 is —NH.
In a preferred embodiment Y 2 is —NH.
In a preferred embodiment R 1 is hydroxyl substituted alkyl.
In a preferred embodiment R 2 and R 3 are hydrogen and v is 1.
In a preferred embodiment Cy 1 is naphthyl or substituted naphthyl.
In a preferred embodiment Cy 2 is an optionally substituted phenyl.
In a preferred embodiment G 1 is —N(H)S(O) 2 —.
In a preferred embodiment, the invention relates to a compound of Formula II:









In a preferred embodiment a compound of Formula I is selected from Table A:








TABLE A





No.
Compound











1














2














3














4














5














6














7














8














9














10














11














12














13














14














15














16














17














18














19














20














21














22














23














24














25














26














27














28














29














30














31














32














33














34














35














36





































































































In a preferred embodiment a compound of Formula I is selected from Table B:






TABLE B



No.
Compound



1A












2A












3A












4A












5A












6A












7A












8A












9A












10A












11A












12A












13A












14A












15A












16A












17A












18A












19A












20A












21A












22A












23A












24A












25A












26A












27A












28A












29A












30A












31A












31B












32A












33A












34A












35A












36A












37A












37B












38A












39A












40A












41A












42A












43A












44A












45A












46A












47A












48A




























































































































The invention further relates to the treatment of autoimmune diseases by the administration of a compound of Formula I. Compounds of Formula I can be useful for the treatment of autoimmune diseases including multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, osteoarthritis, psoriasis, ulcerative colitis, type-1 diabetes, pancreatitis, asthma, idiopathic thrombocytopenia purpura, uveitis, Guillain-Barre syndrome or myasthenia gravis. In a preferred embodiment a patient suffering from type-1 diabetes is treated with a compound of Formula I.
Without being bound by any theory the following postulated to relate the activity of the compounds of Formula I in autoimmune diseases. Human beta-cell apoptosis involves a complex set of signaling cascades initiated by IL-1β, IFN-γ, and TNF-α. IL-1β and TNF-α induce NF-κB expression, while downstream activation of gene expression is thought to occur through nitric oxide (NO) signaling, which both increases the endoplasmic reticulum stress-response pathway and decreases beta-cell function; this pathway works together with IFN-γ-induced STAT-1 signaling to effect beta-cell death. These effects of cytokines are beta cell-specific, and small-molecule suppressors should have little to no effect on other cell types in the pancreas. Small molecules that increase beta-cell survival in the presence of cytokines could be of potential clinical benefit to early-stage type 1 diabetic patients. A number of studies have described small molecules with protective effects in the presence of cytokines; most of these were discovered because of their nonspecific antioxidant or anti-inflammatory effects. Further, small-molecule inhibition of histone deacetylases (HDAC) with suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) can prevent cytokine-induced beta-cell death, presumably by decreasing NF-κB transactivation. Therefore, multiple mechanisms may serve to protect beta cells from cytokine-induced apoptosis. However, these compounds appeared to be somewhat toxic to these cells, as evidenced by a decrease in ATP even without cytokine treatment. Compound-1A inhibited cytokine-induced beta cell apoptosis with an IC 50 of 4.9 μM. Compound-21A exhibited an IC 50 of 770 nM. In addition to acting as a potent suppressor of cytokine-induced beta-cell apoptosis, Compound-21A decreased caspase-3/7 activity in INS-1E cells and restored glucose-stimulated insulin secretion (GSIS), showing improved beta-cell function, to nearly normal levels. It is postulated that this result is not merely due to an increase in cell viability for two reasons. First, the levels of insulin secreted in the presence of low-glucose (2 mM) conditions are nearly the same in all treatments. Second, we have observed other compounds that are capable of increasing ATP levels, but do not restore GSIS in this assay. Thus, Compound-21A is likely suppressing beta-cell apoptosis and restoring important physiological beta-cell function. An important compound for this process will be active in human cells. To that end, we treated dissociated human islets with the same combination of cytokines. Independently, we had developed a dissociated human islet cell-culture system to study beta-cell proliferation. In this system, intact human islets are gently dissociated, and seeded onto 96- or 384-well plates coated with extracellular matrix derived from the HTB-9 human bladder carcinoma cell line. We observed induction of apoptosis after a six-day treatment; these results are consistent with previous reports in human dissociated islets. Six-day treatment induced caspase-3/7 activity by approximately 60%; this effect was abolished by co-incubation with Compound-21A. Because of the use of dissociated islets and a whole-well readout of caspase activity, it cannot be certain that only beta cells are undergoing apoptosis; however, based on previous results in the literature, beta cells may constitute the majority of the apoptotic effect.
The invention further relates to a method of treating a BCL-2 related disease or disorder by administering a compound of Formula I to a subject in need thereof. In one embodiment, the BCL-2 mediated disease or disorder is a cell proliferative disorder. In a preferred embodiment, the cell proliferative disorder is selected from breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In a preferred embodiment, the cell proliferative disorder is selected from the group consisting of papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease and Burkitt's disease.
In one embodiment, the invention relates to a composition comprising a compound of Formula I and another BCL-2 inhibitor. In a preferred embodiment, the composition comprises a compound of Formula I and a second compound selected from ABT-263, ABT-737, gossypol, chelerythrine chloride, apogossypolone, antimycin A, TW-37, HA14-1, obatoclax (GX15-070), ApoG2, NMB and TM12-06. In a more preferred embodiment, the second compound is ABT-737.
In one embodiment, the invention relates to a method of treating a deubiquitinase related disease or disorder comprising administering a compound of Formula I to a subject in need thereof. In one embodiment, the deubiquitinase related disease or disorder is selected from inflammation, cell proliferative disorder, macular degeneration and diabetic retinopathy, Alzheimer's, atherosclerosis, psoriasis, rheumatoid arthritis and endometriosis. In one embodiment the disease or disorder is a cell proliferative disorder, preferably cancer.
In one embodiment, the invention relates to the treatment of a disease or disorder mediated by the abnormal or aberrant expression of one or more gene or gene product selected from Table 1.






TABLE 1



Accession No.
Description



1370202_at
HRAS like suppressor 3, Hrasls3

1369268_at
activating transcription factor 3, Atf3

1372013_at
interferon induced transmembrane protein 1 (predicted),


Ifitm1_predicted

1377156_at
similar to Transcription factor 7-like 2 (HMG box


transcription factor 4) (T-cell-specific transcription factor 4)


(TCF-4) (hTCF-4), LOC683733

1384391_at
retinol dehydrogenase 10 (all-trans), Rdh10

1373923_at
Retinol dehydrogenase 10 (all-trans), Rdh10

1368868_at
A kinase (PRKA) anchor protein (gravin) 12, Akap12

1393351_at
Retinol dehydrogenase 10 (all-trans), Rdh10

1367918_at
fasciculation and elongation protein zeta 1 (zygin I), Fez1

1368869_at
A kinase (PRKA) anchor protein (gravin) 12, Akap12

1388102_at
leukotriene B4 12-hydroxydehydrogenase, Ltb4dh

1392547_at
hypothetical LOC302884, MGC105649

1396327_at
cytochrome P450, family 2, subfamily j, polypeptide 10


(predicted), Cyp2j10_predicted

1368751_at
potassium voltage-gated channel, delayed-rectifier,


subfamily S, member 3, Kcns3

1373309_at
transmembrane protein 86A (predicted),


Tmem86a_predicted

1368375_a_at
interleukin 15, Il15

1370427_at
platelet derived growth factor, alpha, Pdgfa

1388502_at
inositol polyphosphate-5-phosphatase B, Inpp5b

1372111_at
met proto-oncogene /// caveolin, caveolae protein 1 ///


wingless-related MMTV integration site 2 /// ankyrin


repeat, SAM and basic leucine zipper domain containing 1


/// cortactin binding protein 2 /// suppression of


tumorigenicity 7 /// caveolin 2, Asz1 /// Cav1 /// Cav2 ///


Cttnbp2 /// Met /// ST7 /// Wnt2

1374469_at
Transcribed locus, strongly similar to XP_578186.2


PREDICTED: similar to oxidation resistance 1 [ Rattus


norvegicus ], ---

1378679_at
ubiquitin specific peptidase 25, Usp25

1379375_at
Platelet derived growth factor, alpha, Pdgfa

1372302_at
family with sequence similarity 82, member C, Fam82c

1372182_at
phosphofructokinase, platelet, Pfkp

1386979_at
developmentally regulated protein TPO1, Tpo1

1390847_at
transmembrane protein 86A (predicted),


Tmem86a_predicted

1390226_at
similar to hypothetical protein LOC340061 (predicted),


RGD1562552_predicted

1374519_at
dedicator of cytokinesis 7, Dock7

1371033_at
transporter 1, ATP-binding cassette, sub-family B


(MDR/TAP) /// transporter 2, ATP-binding cassette, sub-


family B (MDR/TAP) /// proteosome (prosome, macropain)


subunit, beta type 9 /// similar to butyrophilin-like 8


(predicted) /// RT1 class II, locus Da /// RT1 class II, locus


Db1 /// major histocompatibility complex, class II, DM


beta /// major histocompatibility complex, class II, DM


alpha /// RT1 class II, locus Ba /// RT1 class II, locus Bb ///


RT1 class II, locus DOb /// butyrophilin-like 4 ///


butyrophilin-like 5 /// butyrophilin-like 3 /// Tesb pseudo-


gene, Btnl3 /// Btnl4 /// Btnl5 /// Hla-dma /// Hla-dmb ///


Psmb9 /// RGD1562488_predicted /// RT1-Ba /// RT1-


Bb /// RT1-Da /// RT1-Db1 /// RT1-DOb /// Tap1 ///


Tap2 /// Tesb

1385502_at
Tripartite motif protein 21 (predicted), Trim21_predicted

1371491_at
Notch gene homolog 1 ( Drosophila ), Notch1

1373911_at
periostin, osteoblast specific factor (predicted),


Postn_predicted

1393806_at
MANSC domain containing 1, Mansc1

1373912_at
Ectonucleotide pyrophosphatase/phosphodiesterase 4


(predicted), Enpp4_predicted

1388936_at
cadherin 11, Cdh11

1374404_at
Jun oncogene, Jun

1382074_at
ring finger protein 19B, Rnf19b

1370974_at
vacuolar protein sorting 54 (yeast), Vps54

1368144_at
regulator of G-protein signaling 2, Rgs2

1369788_s_at
Jun oncogene, Jun

1389528_s_at
Jun oncogene, Jun

1369519_at
endothelin 1, Edn1

1395124_at
tryptophanyl-tRNA synthetase, Wars

1376977_at
Prostaglandin E receptor 3 (subtype EP3), Ptger3

1373961_at
similar to 4930453N24Rik protein, MGC95208

1387398_at
protein kinase inhibitor, alpha, Pkia

1389365_at
similar to CG3740-PA, LOC690000

1368856_at
Janus kinase 2, Jak2

1374499_at
CDNA clone MGC: 187619 IMAGE: 8367117, ---

1377893_at
similar to CG3740-PA, LOC690000

1387074_at
regulator of G-protein signaling 2, Rgs2

1372602_at
starch binding domain 1, Stbd1

1395135_at
Williams Beuren syndrome chromosome region 27,


Wbscr27

1398840_at
vesicle-associated membrane protein 5, Vamp5

1387800_at
Fas death domain-associated protein, Daxx

1373761_at
similar to Protein FAM60A (Tera protein), LOC686611

1386998_at
aldolase C, Aldoc

1384457_at
similar to Fbxw17 protein (predicted),


RGD1566133_predicted

1371071_at
guanine nucleotide binding protein, beta 4, Gnb4

1369319_at
ADP-ribosylation factor-like 6 interacting protein 5,


Arl6ip5

1385210_at
dedicator of cytokinesis 5 (predicted), Dock5_predicted

1388469_at
Insulin-like growth factor I mRNA, 3′ end of mRNA, ---

1385961_at
Kruppel-like factor 5, Klf5

1394077_at
Rho family GTPase 3, Rnd3

1376026_at
downstream neighbor of SON, Donson

1368111_at
ankyrin repeat and BTB (POZ) domain containing 2, Abtb2

1375870_a_at
RNA binding motif, single stranded interacting protein 1,


Rbms1

1375739_at
EH-domain containing 4, Ehd4

1383915_at
hypothetical protein LOC686120, LOC686120

1390507_at
interferon stimulated exonuclease 20, Isg20

1373143_at
similar to hypothetical protein FLJ10652, RGD1309621

1379558_at
Similar to zinc finger protein 748 isoform 2, LOC680222

1380110_at
Janus kinase 2, Jak2

1389263_at
retinoic acid induced 14, Rai14

1373412_at
5′-nucleotidase, cytosolic III (predicted), Nt5c3_predicted

1387851_at
phosphotriesterase related, Pter

1387035_a_at
Rho GTPase activating protein 17, Arhgap17

1377663_at
Rho family GTPase 3, Rnd3

1368321_at
early growth response 1, Egr1

1380513_at
RNA polymerase II associated protein 2, Rpap2

1390237_at
translocase of inner mitochondrial membrane 8 homolog a1


(yeast), Timm8a1

1368571_at
CAP-GLY domain containing linker protein 2, Clip2

1372056_at
CKLF-like MARVEL transmembrane domain containing 6,


Cmtm6

1368982_at
protein kinase inhibitor, alpha, Pkia

1388574_at
tryptophanyl-tRNA synthetase, Wars

1373775_at
NEDD8 ultimate buster-1, Nub1

1383013_at
Kruppel-like factor 13, Klf13

1389732_at
Similar to CG4025-PA, LOC679937

1387294_at
SH3-domain binding protein 5 (BTK-associated), Sh3bp5

1385294_at
ets variant gene 6 (TEL oncogene), Etv6

1388027_a_at
reticulon 4, Rtn4

1370428_x_at
RT1 class Ib, locus Aw2 /// RT1 class Ia, locus A1 /// RT1


class Ia, locus A2 /// RT1 class Ib, locus C1 /// RT1 class II,


locus DOa /// RT1 class II, locus Ha /// TAP binding protein


/// discoidin domain receptor family, member 1 /// leucocyte


specific transcript 1 /// Fas death domain-associated


protein /// UDP-Gal:betaGlcNAc beta 1,3-galactosyltrans-


ferase, polypeptide 4 /// synaptic Ras GTPase activating


protein 1 homolog (rat) /// general transcription factor II H,


polypeptide 4 /// procollagen, type XI, alpha 2 /// RT1 class


I, locus Ke4 /// RAB2, member RAS oncogene family-


like /// PHD finger protein 1 /// zinc finger and BTB domain


containing 9 /// POU domain, class 5, transcription factor


1 /// valyl-tRNA synthetase 2-like /// RT1 class I, CE12 ///


RT1 class I, CE1 /// RT1 class I, CE5 /// RT1 class I, A3 ///


WD repeat domain 46 /// MHC class II region expressed


gene KE2 /// zinc finger protein 297 /// lymphotoxin B ///


ATPase, H+ transporting, V1 subunit G isoform 2 /// RT1


class I, CE7 /// transcription factor 19 /// HCR (a-helix


coiled-coil rod homolog) /// psoriasis susceptibility 1


candidate 2 (human) /// corneodesmosin /// RT1 class I,


CE14 /// RT1 class I, CE2 /// RT1 class I, CE4 /// RT1 class


I, CE15 /// RT1 class I, CE13 /// RT1 class I, CE11 /// RT1


class I, CE10 /// RT1 class I, CE3 /// RT1 class I, CE16 ///


similar to corneodesmosin, Atp6v1g2 /// B3galt4 /// Cdsn ///


Col11a2 /// Daxx /// Ddr1 /// Gtf2h4 /// Hcr /// Ke2 ///


LOC682408 /// Lst1 /// Ltb /// Phf1 /// Pou5f1 ///


Psors1c2 /// Rab2l /// RT1-A1 /// RT1-A2 /// RT1-A3 ///


RT1-Aw2 /// RT1-CE1 /// RT1-CE10 /// RT1-CE11 ///


RT1-CE12 ///RT1-CE13 /// RT1-CE14 /// RT1-CE15 ///


RT1-CE16 /// RT1-CE2 /// RT1-CE3 /// RT1-CE4 /// RT1-


CE5 /// RT1-CE7 /// RT1-Cl /// RT1-DOa /// RT1-Ha ///


RT1-Ke4 /// Syngap1 /// Tapbp /// Tcf19 /// Vars2l ///


Wdr46 /// Zbtb9 /// Zfp297

1393217_at
ATP-binding cassette, sub-family G (WHITE), member 3-


like 1, Abcg3l1

1371049_at
dihydropyrimidinase-like 4, Dpysl4

1377387_a_at
Transcribed locus, strongly similar to NP_079738.2


endothelin converting enzyme 2 isoform c


[ Mus musculus ], ---

1385658_at
zinc finger protein 313, Zfp313

1395297_at
similar to hypothetical protein FLJ10652, RGD1309621

1374387_at
ADP-ribosylation factor-like 6 interacting protein 5,


Arl6ip5

1369633_at
chemokine (C—X—C motif) ligand 12, Cxcl12

1389014_at
pre-B-cell colony enhancing factor 1, Pbef1

1387257_at
secretin, Sct

1387646_a_at
Max protein, Max

1370463_x_at
RT1 class I, CE16, RT1-CE16

1381567_at
zinc finger with UFM1-specific peptidase domain, Zufsp

1368181_at
methylenetetrahydrofolate dehydrogenase (NADP+


dependent), methenyltetrahydrofolate cyclohydrolase,


formyltetrahydrofolate synthase, Mthfd1

1374678_at
sema domain, immunoglobulin domain (Ig),


transmembrane domain (TM) and short cytoplasmic


domain, (semaphorin) 4B, Sema4b

1382158_at
zinc finger protein 202, Zfp202

1379450_at
CTTNBP2 N-terminal like (predicted), Cttnbp2nl_predicted

1373831_at
hypothetical protein LOC619574, LOC619574

1377439_at
similar to hypothetical protein FLJ13231 (predicted) ///


hypothetical protein LOC679830, LOC679830 ///


RGD1310081_predicted

1388583_at
chemokine (C—X—C motif) ligand 12, Cxcl12

1372473_at
tight junction protein 1 (predicted), Tjp1_predicted

1376440_at
ring finger protein 139 (predicted), Rnf139_predicted

1382873_at
CTTNBP2 N-terminal like (predicted), Cttnbp2nl_predicted

1373830_at
hypothetical protein LOC619574, LOC619574

1396262_at
pre-B-cell colony enhancing factor 1, Pbef1

1372106_at
EH-domain containing 4, Ehd4

1376082_at
ecotropic viral integration site 1 (predicted), Evi1_predicted

1390036_at
solute carrier family 16 (monocarboxylic acid transporters),


member 6, Slc16a6

1372270_at
dolichyl-phosphate (UDP-N-acetylglucosamine) N-


acetylglucosaminephosphotransferase 1 (GlcNAc-1-P


transferase), Dpagt1

1393234_at
Transcribed locus, strongly similar to NP_001106215.1


Rho guanine nucleotide exchange factor (GEF) 16 [ Mus


musculus ], ---

1379287_at
cylindromatosis (turban tumor syndrome), Cyld

1372695_at
Transcribed locus, strongly similar to NP_081678.1


fibronectin type III domain containing 5


[ Mus musculus ], ---

1382277_at
lymphocyte antigen 96, Ly96

1387265_at
diacylglycerol kinase, gamma, Dgkg

1373669_at
glucosamine-6-phosphate deaminase 2 (predicted),


Gnpda2_predicted

1375216_at
poliovirus receptor-related 2, Pvrl2

1372846_at
cytochrome b, ascorbate dependent 3, Cybasc3

1379561_at
ATPase family, AAA domain containing 1, Atad1

1375177_at
Kruppel-like factor 13, Klf13

1395248_at
ER degradation enhancer, mannosidase alpha-like 1, Edem1

1383829_at
bobby sox homolog ( Drosophila ) (predicted),


Bbx_predicted

1372895_at
similar to RIKEN cDNA 5730469M10, RGD1309676

1380650_at
TRIO and F-actin binding protein, Triobp

1393641_at
B-cell linker, Blnk

1367758_at
alpha-fetoprotein, Afp

1399167_a_at
growth factor receptor bound protein 2-associated protein 1


(predicted), Gab1_predicted

1368274_at
drebrin-like, Dbnl

1389059_at
Transcribed locus, weakly similar to XP_001250613.1


PREDICTED: hypothetical protein [ Bos taurus ], ---

1385411_at
ubiquitin specific protease 43, rCG_32844

1384186_at
ER degradation enhancer, mannosidase alpha-like 1, Edem1

1371624_at
zinc finger CCCH-type containing 7B (predicted),


Zc3h7b_predicted

1375934_at
ring finger protein 128 /// hypothetical protein LOC680663,


LOC680663 /// Rnf128

1389681_at
Transcribed locus, moderately similar to NP_002847.1


poliovirus receptor related 2 isoform alpha precursor


[ Homo sapiens ], ---

1389164_at
hect domain and RLD 3 (predicted), Herc3_predicted

1375582_at
zinc finger homeodomain 4 (predicted), Zfhx4_predicted

1376766_at
formin-like 1 (predicted), Fmnl1_predicted

1386346_at
transmembrane protein 19, Tmem19

1388195_at
CUG triplet repeat, RNA binding protein 2, Cugbp2

1383554_at
ring finger protein 128 /// hypothetical protein LOC680663,


LOC680663 /// Rnf128

1391211_at
Atpase, class VI, type 11C (predicted), Atp11c_predicted

1388071_x_at
RT1 class Ib, locus Aw2, RT1-Aw2

1383662_at
hypothetical protein LOC500956, LOC500956

1388791_at
similar to 2810022L02Rik protein, RGD1309930

1387180_at
interleukin 1 receptor, type II, Il1r2

1373136_at
zinc finger with UFM1-specific peptidase domain, Zufsp

1373913_at
polyribonucleotide nucleotidyltransferase 1, Pnpt1

1369562_at
hippocalcin-like 1, Hpcal1

1371662_at
lysyl-tRNA synthetase, Kars

1392658_at
Similar to transcription elongation factor A 1 isoform 2,


LOC498453

1370972_x_at
RT1 class I, CE5, RT1-CE5

1388409_at
zinc finger CCCH-type containing 7B (predicted),


Zc3h7b_predicted

1372090_at
Max protein, Max

1392958_at
cylindromatosis (turban tumor syndrome), Cyld

1369557_at
caspase 7, Casp7

1376117_at
solute carrier family 44, member 4, Slc44a4

1372533_at
ER degradation enhancer, mannosidase alpha-like 1, Edem1

1394842_at
transmembrane protein 19, Tmem19

1399153_at
RAB5B, member RAS oncogene family (predicted),


Rab5b_predicted

1369197_at
apoptotic peptidase activating factor 1, Apaf1

1391679_at
hypothetical protein LOC691083, LOC691083

1375955_at
zinc finger protein 313, Zfp313

1383551_at
2,3-bisphosphoglycerate mutase, Bpgm

1373501_at
NIMA (never in mitosis gene a)-related expressed kinase 7,


Nek7

1398983_at
mitochondrial ribosomal protein L30 (predicted),


Mrpl30_predicted

1389387_at
similar to Proteasome inhibitor PI31 subunit,


LOC682071 /// LOC689852

1373065_at
protein tyrosine phosphatase, non-receptor type 18, Ptpn18

1377000_at
WAS protein homology region 2 domain containing 1,


Whdc1

1379256_at
similar to RIKEN cDNA 1810030O07 (predicted),


RGD1565685_predicted

1374400_at
EFR3 homolog A ( S. cerevisiae ), Efr3a

1373520_at
CDNA clone IMAGE: 7367270, ---

1372865_at
zinc finger protein 364 (predicted), Zfp364_predicted

1392978_at
solute carrier family 25, member 28, Slc25a28

1372211_at
v-maf musculoaponeurotic fibrosarcoma oncogene family,


protein K (avian), Mafk

1379957_at
schlafen 8, Slfn8

1371774_at
spermidine/spermine N1-acetyl transferase 1, Sat1

1369067_at
nuclear receptor subfamily 4, group A, member 3, Nr4a3

1387131_at
serine (or cysteine) peptidase inhibitor, clade I, member 1,


Serpini1

1375853_at
Similar to CG13957-PA (predicted),


RGD1309995_predicted

1377970_at
poly (ADP-ribose) polymerase family, member 8, Parp8

1388823_at
RAB5B, member RAS oncogene family (predicted),


Rab5b_predicted

1389053_at
Similar to hypothetical protein FLJ20627 (predicted),


RGD1309546_predicted

1371525_at
solute carrier family 12, member 7, Slc12a7

1367710_at
proteasome (prosome, macropain) 28 subunit, beta, Psme2

1391442_at
EH-domain containing 3, Ehd3

1392838_at
similar to CG13957-PA (predicted),


RGD1309995_predicted

1387897_at
2′,3′-cyclic nucleotide 3′ phosphodiesterase, Cnp

1376835_at
Solute carrier family 35, member B2, Slc35b2

1380121_at
NIMA (never in mitosis gene a)-related expressed kinase 7,


Nek7

1388768_at
small G protein signaling modulator 2, Sgsm2

1368476_at
nuclear receptor subfamily 3, group C, member 2, Nr3c2

1367746_a_at
flotillin 2, Flot2

1370946_at
nuclear factor I/X, Nfix

1393389_at
nuclear receptor subfamily 4, group A, member 3, Nr4a3

1368273_at
mitogen-activated protein kinase 6, Mapk6

1388544_at
2,3-bisphosphoglycerate mutase, Bpgm

1375447_at
GTP binding protein 1 (predicted), Gtpbp1_predicted

1399063_at
ZUBR1, Rbaf600

1388233_at
cytokine inducible SH2-containing protein, Cish

1367663_at
proteasome (prosome, macropain) 28 subunit, alpha, Psme1

1380001_at
pinin, Pnn

1390859_at
Nedd4 binding protein 1, N4bp1

1374939_at
cytoplasmic FMR1 interacting protein 2 (predicted),


Cyfip2_predicted

1395336_at
Similar to 2810022L02Rik protein, RGD1309930

1383768_at
ELAV (embryonic lethal, abnormal vision, Drosophila )-


like 2 (Hu antigen B), Elavl2

1368813_at
CCAAT/enhancer binding protein (C/EBP), delta, Cebpd

1376810_at
M-phase phosphoprotein 8 /// poly (ADP-ribose)


polymerase family, member 4, Mphosph8 /// Parp4

1369559_a_at
CD47 antigen (Rh-related antigen, integrin-associated


signal transducer), Cd47

1386922_at
carbonic anhydrase 2, Ca2

1383616_at
interleukin 10 receptor, beta, Il10rb

1368248_at
CDP-diacylglycerol synthase 1, Cds1

1368158_at
sec1 family domain containing 1, Scfd1

1369654_at
protein kinase, AMP-activated, alpha 2 catalytic subunit,


Prkaa2

1392077_at
putative C11orf8 homolog (human), C11orf8h

1376692_at
homeodomain interacting protein kinase 2 (predicted),


Hipk2_predicted

1372179_at
hippocalcin-like 1, Hpcal1

1369590_a_at
DNA-damage inducible transcript 3, Ddit3

1372101_at
phosphatidic acid phosphatase type 2B, Ppap2b

1380867_a_at
pleckstrin homology domain containing, family M (with


RUN domain) member 1, Plekhm1

1371509_at
transforming growth factor beta regulated gene 1, Tbrg1

1375677_at
transducer of ERBB2, 2, Tob2

1391222_at
Nedd4 binding protein 1, N4bp1

1383369_at
tripartite motif-containing 26, Trim26

1367986_at
prostaglandin F2 receptor negative regulator, Ptgfrn

1393359_at
adaptor-related protein complex 3, beta 2 subunit


(predicted), Ap3b2_predicted

1371925_at
ATPase type 13A1 (predicted), Atp13a1_predicted

1373091_at
pleiomorphic adenoma gene-like 2 (predicted),


Plagl2_predicted

1368503_at
GTP cyclohydrolase 1, Gch1

1367733_at
carbonic anhydrase 2, Ca2

1384244_at
hydroxysteroid dehydrogenase like 2, Hsdl2

1389282_at
Integrin alpha 3 (predicted), Itga3_predicted

1380964_at
dystrobrevin alpha (predicted), Dtna_predicted

1393253_at
zinc finger protein 365, Zfp365

1388798_at
ubiquitin-conjugating enzyme E2E 2 (UBC4/5 homolog,


yeast), Ube2e2

1392107_at
strawberry notch homolog 2 ( Drosophila ), Sbno2

1386976_at
CD82 antigen, Cd82

1376737_at
hypothetical protein LOC690243, LOC690243

1372009_at
tyrosyl-tRNA synthetase, Yars

1399161_a_at
type 1 tumor necrosis factor receptor shedding


aminopeptidase regulator, Arts1

1380582_at
colony stimulating factor 1 (macrophage), Csf1

1378293_at
tripartite motif-containing 26, Trim26

1395426_at
ubiquitin-conjugating enzyme E2 variant 1 (predicted),


Ube2v1_predicted

1377759_at
BH3 interacting domain death agonist, Bid

1383679_at
ring finger protein 31 (predicted), Rnf31_predicted

1368716_at
protein phosphatase 1, regulatory (inhibitor) subunit 14c,


Ppp1r14c

1392518_at
Similar to Protein C22orf5, RGD1306591

1386926_at
acyl-CoA synthetase long-chain family member 5, Acsl5

1375006_at
CDNA clone IMAGE: 7318427, ---

1389913_at
leucine rich repeat (in FLII) interacting protein 1, Lrrfip1

1373588_at
FERM domain containing 8, Frmd8

1382710_at
Ectodermal-neural cortex 1, Enc1

1371550_at
TSC22 domain family, member 4, Tsc22d4

1368679_a_at
Yamaguchi sarcoma viral (v-yes-1) oncogene homolog,


Lyn

1389331_at
CDNA clone IMAGE: 7313169, ---

1381570_at
T-cell activation NFKB-like protein, Ta-nfkbh

1367696_at
interferon induced transmembrane protein 2, Ifitm2

1396278_at
sorting nexin 11, Snx11

1393703_at
human immunodeficiency virus type I enhancer binding


protein 3 (predicted), Hivep3_predicted

1379460_at
SH3-domain GRB2-like (endophilin) interacting protein 1,


Sgip1

1371005_at
ATP-binding cassette, sub-family C (CFTR/MRP), member


1, Abcc1

1368928_at
tripartite motif-containing 3, Trim3

1371531_at
similar to mammalian retrotransposon derived 8b,


LOC678880

1368674_at
liver glycogen phosphorylase, Pygl

1368896_at
MAD homolog 7 ( Drosophila ), Smad7

1387221_at
GTP cyclohydrolase 1, Gch1

1385361_at
ATPase, class V, type 10A, Atp10a

1376174_at
serine (or cysteine) peptidase inhibitor, clade A (alpha-1


antiproteinase, antitrypsin), member 11, Serpina11

1390423_at
MYC binding protein 2, Mycbp2

1372869_at
similar to isopentenyl diphosphate delta-isomerase type 2


(predicted) /// similar to Nucleolar GTP-binding protein 1


(Chronic renal failure gene protein) (GTP-binding protein


NGB), LOC689842 /// RGD1560805_predicted

1391897_at
RAB GTPase activating protein 1-like, Rabgap1l

1371825_at
small nuclear RNA activating complex, polypeptide 2,


Snapc2

1382775_at
ryanodine receptor 2, cardiac, Ryr2

1385758_at
SAPS domain family, member 1 (predicted),


Saps1_predicted

1382029_at
similar to CDNA sequence BC017647 (predicted),


RGD1566149_predicted

1371528_at
FK506 binding protein 8, Fkbp8

1367938_at
UDP-glucose dehydrogenase, Ugdh

1371659_at
ras homolog gene family, member C (predicted),


Rhoc_predicted

1371731_at
Similar to Coatomer gamma-2 subunit (Gamma-2 coat


protein) (Gamma-2 COP) (predicted),


RGD1566215_predicted

1373748_at
PDZ domain containing RING finger 3 (predicted),


Pdzrn3_predicted

1374035_at
rad and gem related GTP binding protein 2, Rem2

1368983_at
heparin-binding EGF-like growth factor, Hbegf

1375861_at
nucleosome assembly protein 1-like 5, Nap1l5

1394022_at
inhibitor of DNA binding 4, Id4

1370606_at
purinergic receptor P2Y, G-protein coupled 1, P2ry1

1368641_at
wingless-related MMTV integration site 4, Wnt4

1382351_at
GTP binding protein (gene overexpressed in skeletal


muscle) (predicted), Gem_predicted

1368998_at
NK6 transcription factor related, locus 1 ( Drosophila ),


Nkx6-1

1375120_at
inhibitor of DNA binding 4, Id4

1384262_at
protein phosphatase 1, regulatory (inhibitor) subunit 3B,


Ppp1r3b

1384448_at
similar to RIKEN cDNA 1700045119 (predicted),


RGD1565844_predicted

1371091_at
insulin receptor substrate 2, Irs2

1378899_at
solute carrier family 35, member D3 (predicted),


Slc35d3_predicted

1392590_at
Rho GTPase activating protein 24, Arhgap24

1376901_a_at
Similar to Hypothetical protein 6330514E13 (predicted),


RGD1559693_predicted

1367948_a_at
kinase insert domain protein receptor, Kdr

1378925_at
cAMP responsive element modulator, Crem

1379419_at
transmembrane protein 34, Tmem34

1372308_at
CDNA clone IMAGE: 7366335, ---

1379724_at
pleckstrin homology-like domain, family B, member 2,


Phldb2

1390403_at
family with sequence similarity 43, member A, Fam43a

1393550_at
cAMP responsive element modulator, Crem

1382312_at
AT rich interactive domain 5B (Mrf1 like) (predicted),


Arid5b_predicted

1367977_at
synuclein, alpha, Snca

1388108_at
ELOVL family member 6, elongation of long chain fatty


acids (yeast), Elovl6

1390148_a_at
zinc finger protein 395 (predicted), Zfp395_predicted

1369737_at
cAMP responsive element modulator, Crem

1370478_at
myosin XVI, Myo16

1390127_at
DIX domain containing 1, Dixdc1

1392183_at
homeobox C9, Hoxc9

1373786_at
zinc finger protein 703, Zfp703

1369770_at
somatostatin receptor 1, Sstr1

1370942_at
RAS p21 protein activator 3, Rasa3

1397587_at
Histone deacetylase 5, Hdac5

1387455_a_at
very low density lipoprotein receptor, Vldlr

1376265_at
six transmembrane epithelial antigen of the prostate 2,


Steap2

1387947_at
v-maf musculoaponeurotic fibrosarcoma oncogene family,


protein B (avian), Mafb

1385374_at
Transcribed locus, strongly similar to NP_062067.2


thyrotroph embryonic factor [ Rattus norvegicus ], ---

1397729_x_at
similar to RIKEN cDNA 1600029D21, LOC363060

1381605_at
ubiquitin specific protease 13 (isopeptidase T-3)


(predicted), Usp13_predicted

1369564_at
rad and gem related GTP binding protein 2, Rem2

1387477_at
potassium channel, subfamily K, member 12, Kcnk12

1395223_at
kelch domain containing 8B, Klhdc8b

1372548_at
Transcribed locus, strongly similar to NP_066940.1


cryptochrome 2 (photolyase-like) [ Homo sapiens ], ---

1385387_at
NK2 transcription factor related, locus 2 ( Drosophila )


(predicted), Nkx2-2_predicted

1385491_at
PNMA-like 2, Pnmal2

1367805_at
glutaminase, Gls

1374594_at
similar to RIKEN cDNA 1600029D21, LOC363060

1373179_at
similar to H43E16.1, LOC689994

1370474_at
thyroid hormone receptor beta, Thrb

1367627_at
glycine amidinotransferase (L-arginine:glycine


amidinotransferase), Gatm

1383721_at
frizzled homolog 8 ( Drosophila ), Fzd8

1375043_at
FBJ osteosarcoma oncogene, Fos

1389611_at
very low density lipoprotein receptor, Vldlr

1393691_at
hypothetical protein LOC688273, LOC688273

1377404_at
stanniocalcin 1, Stc1

1379863_at
potassium voltage gated channel, Shal-related family,


member 2, Kcnd2

1368782_at
somatostatin receptor 2, Sstr2

1391117_at
PNMA-like 2, Pnmal2

1387968_at
solute carrier family 6 (neurotransmitter transporter),


member 15, Slc6a15

1392663_at
similar to hypothetical protein FLJ13188 (predicted),


RGD1305500_predicted

1370058_at
neurofilament, light polypeptide, Nefl

1388395_at
G0/G1 switch gene 2, G0s2

1377940_at
family with sequence similarity 101, member B, Fam101b

1369098_at
very low density lipoprotein receptor, Vldlr

1387370_at
tropomodulin 1, Tmod1

1385663_at
ubiquitin specific protease 13 (isopeptidase T-3)


(predicted), Usp13_predicted

1370111_at
potassium intermediate/small conductance calcium-


activated channel, subfamily N, member 2, Kcnn2

1382924_at
pantothenate kinase 1 (predicted), Pank1_predicted

1367857_at
fatty acid desaturase 1, Fads1

1381190_at
LIM domain only protein 7, LMO7

1373336_at
G protein-coupled receptor, family C, group 5, member B


(predicted), Gprc5b_predicted

1369752_a_at
calcium/calmodulin-dependent protein kinase IV, Camk4

1390339_at
Ubiquitin specific protease 13 (isopeptidase T-3)


(predicted), Usp13_predicted

1383390_at
family with sequence similarity 123A, Fam123a

1383826_at
Rab40b, member RAS oncogene family (predicted),


Rab40b_predicted

1379374_at
plasticity related gene 1, Lppr4

1385555_at
family with sequence similarity 101, member A, Fam101a

1398431_at
carbonic anhydrase 8, Car8

1387212_at
basic helix-loop-helix domain containing, class B, 8,


Bhlhb8

1389911_at
meteorin, glial cell differentiation regulator-like, Metrnl

1393615_at
similar to DEP domain containing 6 (predicted),


RGD1561030_predicted

1398732_at
hypothetical protein LOC688273, LOC688273

1387599_a_at
NAD(P)H dehydrogenase, quinone 1, Nqo1

1374906_at
ring finger protein 113A1, Rnf113a1

1374354_at
PHD finger protein 19 (predicted), Phf19_predicted

1372754_at
adaptor protein, phosphotyrosine interaction, PH domain


and leucine zipper containing 2, Appl2

1384415_at
SRY-box containing gene 7 (predicted), Sox7_predicted

1379626_at
Special AT-rich sequence binding protein 1, Satb1

1373985_at
Similar to KIAA1183 protein (predicted),


RGD1560435_predicted

1373108_at
protein phosphatase 1, regulatory (inhibitor) subunit 3C,


Ppp1r3c

1375726_at
LIM domain only 7, Lmo7

1384869_at
abhydrolase domain containing 7 (predicted),


Abhd7_predicted

1390097_at
TSPY-like 4, Tspyl4

1369792_at
G protein-coupled receptor 6, Gpr6

1369753_at
calcium/calmodulin-dependent protein kinase IV, Camk4

1390443_at
similar to DNA segment, Chr 16, ERATO Doi 472,


expressed (predicted), RGD1563888_predicted

1369095_at
protein phosphatase 1, regulatory (inhibitor) subunit 9A,


Ppp1r9a

1376649_at
SNF1-like kinase 2 (predicted), Snf1lk2_predicted

1376687_at
ubiquitin specific peptdiase 1, Usp1

1369738_s_at
cAMP responsive element modulator, Crem

1389514_at
leucine rich repeat and Ig domain containing 1, Lingo1

1380305_at
NOD3-like protein, nod3l

1387349_at
short stature homeobox 2, Shox2

1370317_at
asparagine-linked glycosylation 10 homolog B (yeast,


alpha-1,2-glucosyltransferase), Alg10b

1388078_a_at
amiloride-sensitive cation channel 2, neuronal, Accn2

1393952_at
coiled-coil domain containing 68, Ccdc68

1391601_at
leucine rich repeat protein 2, neuronal (predicted),


Lrrn2_predicted

1385036_at
synuclein, alpha interacting protein (synphilin) (predicted),


Sncaip_predicted

1383573_at
teashirt zinc finger family member 1, Tshz1

1376958_at
Similar to serine (or cysteine) proteinase inhibitor, clade B,


member 9 (predicted), RGD1562844_predicted

1372447_at
Fibroblast growth factor receptor 1, Fgfr1

1385636_at
frizzled homolog 3 ( Drosophila ), Fzd3

1370122_at
RAB27B, member RAS oncogene family, Rab27b

1371045_at
amiloride-sensitive cation channel 2, neuronal, Accn2

1377506_at
LAG1 homolog, ceramide synthase 1 /// growth


differentiation factor 1 (predicted), Gdf1_predicted ///


Lass1

1379292_at
Similar to 5730420B22Rik protein (predicted),


RGD1306755_predicted

1372457_at
mitochondrial tumor suppressor 1, Mtus1

1374706_at
Growth differentiation factor 11, Gdf11

1379703_at
DENN/MADD domain containing 3, Dennd3

1383483_at
RAB9B, member RAS oncogene family (predicted),


Rab9b_predicted

1382868_at
sema domain, transmembrane domain (TM), and


cytoplasmic domain, (semaphorin) 6A (predicted),


Sema6a_predicted

1368825_at
short stature homeobox 2, Shox2

1369954_at
isocitrate dehydrogenase 1 (NADP+), soluble, Idh1

1388718_at
tropomodulin 1, Tmod1

1370218_at
lactate dehydrogenase B, Ldhb

1393307_at
Phosphatase and actin regulator 3, Phactr3

1372365_at
Ras and Rab interactor 2 (predicted), Rin2_predicted

1386960_at
solute carrier family 37 (glucose-6-phosphate transporter),


member 4, Slc37a4

1372117_at
myotubularin related protein 10, Mtmr10

1383443_at
similar to UPF0308 protein C9orf21, LOC498685

1374236_at
leucine-rich repeat LGI family, member 2 (predicted),


Lgi2_predicted

1391194_at
Sal-like 1 ( Drosophila ) (predicted), Sall1_predicted

1398606_at
golgi integral membrane protein 4, Golim4

1390289_at
FIG4 homolog ( S. cerevisiae ), Fig4

1371421_at
similar to 3-oxoacid CoA transferase 1, LOC678860

1379257_at
erythrocyte protein band 4.1-like 4a (predicted),


Epb4.1l4a_predicted

1370941_at
platelet derived growth factor receptor, alpha polypeptide,


Pdgfra

1392739_a_at
endonuclease/exonuclease/phosphatase family domain


containing 1, Eepd1

1376187_at
Solute carrier family 35 (UDP-glucuronic acid/UDP-N-


acetylgalactosamine dual transporter), member D1


(predicted), Slc35d1_predicted

1397267_at
RIM binding protein 2, Rimbp2

1384009_at
nuclear prelamin A recognition factor, Narf

1384238_at
tweety homolog 2 ( Drosophila ), Ttyh2

1385175_at
homeo box B13 (predicted), Hoxb13_predicted

1389054_at
similar to RIKEN cDNA 0610040J01, LOC498368

1396492_at
Nfat activating molecule with ITAM motif 1, Nfam1

1375909_at
similar to glutathione transferase GSTM7-7, MGC108896

1392040_at
spindle assembly 6 homolog ( C. elegans ) (predicted),


Sass6_predicted

1375353_at
Cdk5 and Abl enzyme substrate 1 (predicted),


Cables1_predicted

1390993_at
phenazine biosynthesis-like protein domain containing,


Pbld

1372069_at
ankyrin repeat domain 15, Ankrd15

1389787_at
PTK7 protein tyrosine kinase 7 (predicted), Ptk7_predicted

1390168_a_at
DPH4 homolog (JJJ3, S. cerevisiae ), Dph4

1397861_at
V-set and transmembrane domain containing 2B, Vstm2b

1368160_at
insulin-like growth factor binding protein 1, Igfbp1

1382478_at
BTB (POZ) domain containing 3 (predicted),


Btbd3_predicted

1389003_at
Rho-related BTB domain containing 3 (predicted),


Rhobtb3_predicted

1370407_at
prenylcysteine oxidase 1, Pcyox1

1384331_at
sulfiredoxin 1 homolog ( S. cerevisiae ), Srxn1

1392789_at
solute carrier family 25, member 36, Slc25a36

1390481_a_at
ubiquitin-conjugating enzyme E2T (putative) (predicted),


Ube2t_predicted

1377599_at
lipin 1, Lpin1

1369686_at
doublecortin-like kinase 1, Dclk1

1370106_at
fibroblast growth factor 18, Fgf18

1390050_at
similar to Golgi phosphoprotein 2 (Golgi membrane protein


GP73), LOC680692 /// LOC682869

1390647_at
putative homeodomain transcription factor 2 (predicted),


Phtf2_predicted

1371131_a_at
thioredoxin interacting protein, Txnip

1388703_at
endothelial cell adhesion molecule, Esam

1381969_at
similar to Recombining binding protein suppressor of


hairless (J kappa-recombination signal binding protein)


(RBP-J kappa), LOC679028

1379541_at
transmembrane and tetratricopeptide repeat containing 4,


Tmtc4

1373398_at
Tripartite motif protein 37 (predicted), Trim37_predicted

1367806_at
glutaminase, Gls

1388973_at
procollagen, type IX, alpha 1, Col9a1

1385321_at
similar to Protein arginine N-methyltransferase 4


(Heterogeneous nuclear ribonucleoprotein


methyltransferase-like protein 4), LOC688502

1370416_at
Max dimerization protein 3, Mxd3

1376873_at
cerebellin 1, Cbln1

1375423_at
hypothetical protein LOC689959, LOC689959

1373291_at
deleted in liver cancer 1, Dlc1

1377729_at
elongation of very long chain fatty acids (FEN1/Elo2,


SUR4/Elo3, yeast)-like 4 (predicted), Elovl4_predicted

1392943_at
membrane bound O-acyltransferase domain containing 2,


Mboat2

1367899_at
coagulation factor II (thrombin) receptor, F2r

1389067_at
solute carrier organic anion transporter family, member


4a1, Slco4a1

1370158_at
myosin, heavy polypeptide 10, non-muscle, Myh10

1396112_at
myotubularin related protein 10, Mtmr10

1387503_at
carboxypeptidase N, polypeptide 1, Cpn1

1389632_at
Rho-related BTB domain containing 1 (predicted),


Rhobtb1_predicted

1386129_at
inhibitor of growth family, member 2, Ing2

1377669_at
RAB27A, member RAS oncogene family, Rab27a

1379817_at
purine-rich element binding protein G (predicted),


Purg_predicted

1379302_at
recombining binding protein suppressor of hairless


( Drosophila ) (predicted) /// similar to Recombining binding


protein suppressor of hairless (J kappa-recombination signal


binding protein) (RBP-J kappa), LOC679028 ///


Rbpsuh_predicted

1374333_at
similar to RIKEN cDNA 1110007C09 (predicted),


RGD1306058_predicted

1383433_at
kelch-like 23 ( Drosophila ) (predicted), Klhl23_predicted

1370059_at
neurofilament, light polypeptide, Nefl

1368174_at
EGL nine homolog 3 ( C. elegans ), Egln3

1391871_at
StAR-related lipid transfer (START) domain containing 13,


Stard13

1395160_at
proline-rich transmembrane protein 3, Prrt3

1367869_at
oxidation resistance 1, Oxr1

1381974_at
BTB (POZ) domain containing 3 (predicted),


Btbd3_predicted

1393101_at
F-box and leucine-rich repeat protein 10, Fbxl10

1374846_at
hexamethylene bis-acetamide inducible 1, Hexim1

1374748_at
serine hydroxymethyltransferase 1 (soluble), Shmt1

1375877_at
synaptotagmin IV, Syt4

1398296_at
glycerophosphodiester phosphodiesterase 1, Gde1

1371963_at
propionyl-coenzyme A carboxylase, alpha polypeptide,


Pcca

1398484_at
similar to TBC1 domain family, member 8 (with GRAM


domain); vascular Rab-GAP/TBC-containing (predicted),


RGD1308221_predicted

1391077_at
claspin homolog ( Xenopus laevis ) (predicted),


Clspn_predicted

1369735_at
growth arrest specific 6, Gas6

1390406_at
Rho GTPase activating protein 18 (predicted),


Arhgap18_predicted

1384516_at
Metal response element binding transcription factor 2, Mtf2

1370247_a_at
peripheral myelin protein 22, Pmp22

1387623_at
stanniocalcin 1, Stc1

1386577_at
kelch-like 23 ( Drosophila ) (predicted), Klhl23_predicted

1370237_at
hydroxyacyl-Coenzyme A dehydrogenase, Hadh

1385302_at
glycerophosphodiester phosphodiesterase domain


containing 1 (predicted), Gdpd1_predicted

1373959_at
protein phosphatase 2 (formerly 2A), regulatory subunit A


(PR 65), beta isoform, Ppp2r1b

1398398_at
homeo box A10, Hoxa10

1383601_at
CDNA clone MGC: 188768 IMAGE: 9101044, ---

1370268_at
potassium voltage-gated channel, shaker-related subfamily,


member 5, Kcna5

1389549_at
proline synthetase co-transcribed (predicted),


Prosc_predicted

1374160_at
transmembrane and coiled-coil domains 2, Tmcc2

1374890_at
Transcribed locus, strongly similar to NP_005656.4


ecotropic viral integration site 5 [ Homo sapiens ], ---

1390228_at
amine oxidase, flavin containing 1 (predicted),


Aof1_predicted

1378235_at
glycerophosphodiester phosphodiesterase domain


containing 1 (predicted), Gdpd1_predicted

1392406_at
IAP promoted placental gene (predicted), Ipp_predicted

1371983_at
Josephin domain containing 1, Josd1

1389791_at
ceroid-lipofuscinosis, neuronal 8, Cln8

1392165_at
inhibitor of growth family, member 2, Ing2

1387805_at
BCL2/adenovirus E1B 19 kDa-interacting protein 3, Bnip3

1374491_at
CKLF-like MARVEL transmembrane domain containing 8,


Cmtm8

1387310_at
ATPase, Ca++ transporting, type 2C, member 2, Atp2c2

1384852_at
RAB27A, member RAS oncogene family, Rab27a

1368924_at
growth hormone receptor, Ghr

1368964_at
leucine rich repeat protein 3, neuronal, Lrrn3

1376213_at
similar to Rap2-binding protein 9, MGC124740

1369696_at
Ras-related GTP binding B, RragB

1373043_at
stromal cell-derived factor 2-like 1 (predicted),


Sdf2l1_predicted

1372782_a_at
adenosine monophosphate deaminase 2 (isoform L),


Ampd2

1383382_at
similar to jumonji protein, LOC681740

1390028_at
dual-specificity tyrosine-(Y)-phosphorylation regulated


kinase 2 (predicted), Dyrk2_predicted

1383326_a_at
programmed cell death 4, Pdcd4

1399005_at
protein phosphatase 2, regulatory subunit B (B56), alpha


isoform (predicted), Ppp2r5a_predicted

1389301_at
similar to muscleblind-like 2 isoform 1, LOC680445

1368440_at
solute carrier family 3, member 1, Slc3a1

1377869_at
CCR4 carbon catabolite repression 4-like B ( S. cerevisiae ),


Ccrn4lb

1372093_at
Max interacting protein 1, Mxi1

1370548_at
solute carrier family 16 (monocarboxylic acid transporters),


member 10, Slc16a10

1390249_at
septin 14, Sept14

1388919_at
zinc finger protein 541, Zfp541

1397173_at
similar to Serine/threonine-protein kinase WNK3 (Protein


kinase, lysine-deficient 3) (predicted),


RGD1563131_predicted

1382814_at
odd Oz/ten-m homolog 3 ( Drosophila ) (predicted),


Odz3_predicted

1373659_at
hypothetical protein LOC688257, LOC688257

1391906_at
cortistatin /// kinesin family member 1B, Cort /// Kif1b

1378430_at
monooxygenase, DBH-like 1, Moxd1

1373656_at
protein phosphatase 1, regulatory subunit 3D, Ppp1r3d

1376758_at
inhibitor of growth family, member 1, Ing1

1375059_at
zinc finger protein 652, Zfp652

1374957_at
similar to ribosomal protein L27a (predicted),


RGD1560633_predicted

1384877_at
aquaporin 11, Aqp11

1370054_at
cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4),


Cdkn2c

1367755_at
cysteine dioxygenase 1, cytosolic, Cdo1

1383328_x_at
programmed cell death 4, Pdcd4

1379440_at
follistatin-like 3, Fstl3

1395623_at
glutaminyl-peptide cyclotransferase-like (predicted),


Qpctl_predicted

1370036_at
sulfite oxidase, Suox

1369670_at
Cd200 antigen, Cd200

1392953_at
protein tyrosine phosphatase-like (proline instead of


catalytic arginine), member a, Ptpla

1383534_at
Transcribed locus, moderately similar to XP_001479249.1


PREDICTED: similar to zinc finger protein 605 [ Mus


musculus ], ---

1384885_at
tektin 2, Tekt2

1368122_at
ring finger protein 103, Rnf103

1373625_at
serine hydroxymethyltransferase 1 (soluble), Shmt1

1388856_at
kit ligand, Kitl

1367702_at
acyl-Coenzyme A dehydrogenase, medium chain, Acadm

1374625_at
hairy and enhancer of split 6 ( Drosophila ), Hes6

1369785_at
phosphoribosyl pyrophosphate amidotransferase, Ppat

1385829_at
obscurin-like 1, Obsl1

1383962_at
SIVA1, apoptosis-inducing factor, Siva1

1383666_at
peptidyl-tRNA hydrolase 1 homolog ( S. cerevisiae )


(predicted), Ptrh1_predicted

1373345_at
adhesion molecule with Ig like domain 2, Amigo2

1391919_at
transcription elongation regulator 1-like, Tcerg1l

1384392_at
cytochrome P450, family 26, subfamily b, polypeptide 1,


Cyp26b1

1374540_at
cell division cycle associated 7, Cdca7

1385503_at
similar to O-acetyltransferase, LOC678772

1393439_a_at
progressive ankylosis, Ank

1374030_at
similar to KIAA0999 protein, LOC684112

1377995_at
integrin alpha FG-GAP repeat containing 3, Itfg3

1393218_at
similar to 2410024A21Rik protein, RGD1304878

1383722_at
proline synthetase co-transcribed (predicted),


Prosc_predicted

1374235_at
regulator of calcineurin 2, Rcan2

1372248_at
sestrin 1 (predicted), Sesn1_predicted

1387670_at
glycerol-3-phosphate dehydrogenase 2, mitochondrial,


Gpd2

1387032_at
cholecystokinin, Cck

1395269_s_at
gamma-aminobutyric acid (GABA-A) receptor, subunit


delta, Gabrd

1387662_at
synaptotagmin IV, Syt4

1369010_at
CHK2 checkpoint homolog ( S. pombe ), Chek2

1393347_at
integrin alpha L, Itgal

1376963_at
dual-specificity tyrosine-(Y)-phosphorylation regulated


kinase 2 (predicted), Dyrk2_predicted

1371763_at
similar to RIKEN cDNA 4931406C07, RGD1309534

1386978_at
BCL2/adenovirus E1B interacting protein 3-like, Bnip3l

1370814_at
dehydrogenase/reductase (SDR family) member 4, Dhrs4

1369457_a_at
synaptotagmin-like 4, Sytl4

1367853_at
solute carrier family 12 (sodium/potassium/chloride


transporters), member 2, Slc12a2

1392079_at
A kinase (PRKA) anchor protein 7, Akap7

1395968_at
similar to Myosin-15 (Myosin XV) (Unconventional


myosin-15), LOC688264

1369412_a_at
solute carrier family 19 (sodium/hydrogen exchanger),


member 1, Slc19a1

1381515_at
CDNA clone IMAGE: 7123062, ---

1370120_at
follistatin-like 3, Fstl3

1379258_at
kelch-like 5 ( Drosophila ), Klhl5

1373734_at
Solute carrier organic anion transporter family, member


3a1, Slco3a1

1389084_at
Transcribed locus, weakly similar to XP_001480799.1


PREDICTED: hypothetical protein [ Mus musculus ], ---

1393109_at
CDNA clone IMAGE: 7302574, ---

1373480_at
heat shock 70 kDa protein 12A (predicted),


Hspa12a_predicted

1367791_at
receptor (calcitonin) activity modifying protein 1, Ramp1

1396317_at
cell cycle progression 1 (predicted), Ccpg1_predicted

1387521_at
programmed cell death 4, Pdcd4

1374976_a_at
Sterol O-acyltransferase 1, Soat1

1384210_at
MICAL C-terminal like, Micalcl

1369000_at
neurotrophic tyrosine kinase, receptor, type 1, Ntrk1

1389876_at
Calcium/calmodulin-dependent protein kinase II inhibitor 1,


Camk2n1

1371679_at
synaptopodin 2, Synpo2

1383444_at
solute carrier family 24 (sodium/potassium/calcium


exchanger), member 2, Slc24a2

1379108_at
Similar to ADP-ribosylation factor guanine nucleotide


factor 6 isoform a (predicted), RGD1559968_predicted

1387271_at
phytanoyl-CoA hydroxylase, Phyh

1390207_at
large tumor suppressor 2 (predicted), Lats2_predicted

1368514_at
monoamine oxidase B, Maob

1393196_at
kelch-like 23 ( Drosophila ) (predicted), Klhl23_predicted

1379608_at
RGD1560010 (predicted), RGD1560010_predieted

1388958_a_at
solute carrier family 2 (facilitated glucose transporter),


member 4, Slc2a4

1383300_at
kelch-like 24 ( Drosophila ), Klhl24

1374557_at
G protein-coupled receptor 177, Gpr177

1371113_a_at
transferrin receptor, Tfrc

1393848_at
ribonucleotide reductase M2, Rrm2




























































































































































































































































































































































































































































































































































































































































































































































































































































































































The invention further relates to a compound of Formula II:

Wherein e is 0, 1, 2, 3 or 4;

R 50 is —C(O)R 54 , —C(O)N(H)R 54 , —C(S)R 54 , —C(S)N(H)R 54 ;
wherein R 54 is alkyl or substituted alkyl, —(CH 2 ) 2 O(CH 2 CH 2 O) f CH 2 CH 2 NH 2 , —(CH 2 ) 2 —O—(CH 2 CH 2 ) g NH 2 , —(CH 2 CH 2 ) g NH 2 , —(CH 2 ) 2 —O—(CH 2 CH 2 O) f CH 2 CH 2 SH, —(CH 2 ) 2 —O—(CH 2 CH 2 ) g SH, —(CH 2 CH 2 ) g OH, —(CH 2 ) 2 —O—(CH 2 CH 2 O) f CH 2 CH 2 OH, —(CH 2 ) 2 —O—(CH 2 CH 2 ) g OH, —(CH 2 CH 2 ) g OH;
Wherein each f and g is independently, an integer between 0 and 500, preferably, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 17.
Each R 51 , R 52 and R 53 is independently hydrogen, halogen, —OR 10 , —SR 10 , —NR 10 R 11 , —C(O)R 10 , —C(O)OR 10 , —C(O)NR 10 R 11 , —N(R 10 )C(O)R 11 , —CF 3 , —CN, —NO 2 , —N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl.

The compounds of Formula II can be immobilized onto a surface or a bead through the R 50 group. The immobilized compounds can be used to identify proteins, genes or DNA that can interact with the immobilized compound.
In a preferred embodiment, the invention relates to a compound of Formula II having the structure:









DEFINITIONS
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.
The term “aliphatic group” or “aliphatic” refers to a non-aromatic moiety that may be saturated (e.g. single bond) or contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic, contain carbon, hydrogen or, optionally, one or more heteroatoms and may be substituted or unsubstituted. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and substituted or unsubstituted cycloalkyl groups as described herein.
The term “acyl” refers to a carbonyl substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, or heteroaryl. For example, acyl includes groups such as (C 1 -C 6 )alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C 3 -C 6 )cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions. When indicated as being “optionally substituted”, the acyl group may be unsubstituted or optionally substituted with one or more substituents (typically, one to three substituents) independently selected from the group of substituents listed below in the definition for “substituted” or the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be substituted as described above in the preferred and more preferred list of substituents, respectively.
The term “alkyl” is intended to include branched, straight chain and cyclic, substituted or unsubstituted saturated aliphatic hydrocarbon radicals/groups having the specified number of carbons. Preferred alkyl groups comprise about 1 to about 24 carbon atoms (“C 1 -C 24 ”) preferably about 7 to about 24 carbon atoms (“C 7 -C 24 ”), preferably about 8 to about 24 carbon atoms (“C 8 -C 24 ”), preferably about 9 to about 24 carbon atoms (“C 9 -C 24 ”). Other preferred alkyl groups comprise at about 1 to about 8 carbon atoms (“C 1 -C 8 ”) such as about 1 to about 6 carbon atoms (“C 1 -C 6 ”), or such as about 1 to about 3 carbon atoms (“C 1 -C 3 ”). Examples of C 1 -C 6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl and n-hexyl radicals.
The term “alkenyl” refers to linear or branched radicals having at least one carbon-carbon double bond. Such radicals preferably contain from about two to about twenty-four carbon atoms (“C 2 -C 24 ”) preferably about 7 to about 24 carbon atoms (“C 7 -C 24 ”), preferably about 8 to about 24 carbon atoms (“C 8 -C 24 ”), and preferably about 9 to about 24 carbon atoms (“C 9 -C 24 ”). Other preferred alkenyl radicals are “lower alkenyl” radicals having two to about ten carbon atoms (“C 2 -C 10 ”) such as ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl. Preferred lower alkenyl radicals include 2 to about 6 carbon atoms (“C 2 -C 6 ”). The terms “alkenyl”, and “lower alkenyl”, embrace radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations.
The term “alkynyl” refers to linear or branched radicals having at least one carbon-carbon triple bond. Such radicals preferably contain from about two to about twenty-four carbon atoms (“C 2 -C 24 ”) preferably about 7 to about 24 carbon atoms (“C 7 -C 24 ”), preferably about 8 to about 24 carbon atoms (“C 8 -C 24 ”), and preferably about 9 to about 24 carbon atoms (“C 9 -C 24 ”). Other preferred alkynyl radicals are “lower alkynyl” radicals having two to about ten carbon atoms such as propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl and 1-pentynyl. Preferred lower alkynyl radicals include 2 to about 6 carbon atoms (“C 2 -C 6 ”).
The term “cycloalkyl” refers to saturated carbocyclic radicals having three to about twelve carbon atoms (“C 3 -C 12 ”). The term “cycloalkyl” embraces saturated carbocyclic radicals having three to about twelve carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term “cycloalkenyl” refers to partially unsaturated carbocyclic radicals having three to twelve carbon atoms. Cycloalkenyl radicals that are partially unsaturated carbocyclic radicals that contain two double bonds (that may or may not be conjugated) can be called “cycloalkyldienyl”. More preferred cycloalkenyl radicals are “lower cycloalkenyl” radicals having four to about eight carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl and cyclohexenyl.
The term “alkylene,” as used herein, refers to a divalent group derived from a straight chain or branched saturated hydrocarbon chain having the specified number of carbons atoms. Examples of alkylene groups include, but are not limited to, ethylene, propylene, butylene, 3-methyl-pentylene, and 5-ethyl-hexylene.
The term “alkenylene,” as used herein, denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon double bond. Alkenylene groups include, but are not limited to, for example, ethenylene, 2-propenylene, 2-butenylene, 1-methyl-2-buten-1-ylene, and the like.
The term “alkynylene,” as used herein, denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon triple bond. Representative alkynylene groups include, but are not limited to, for example, propynylene, 1-butynylene, 2-methyl-3-hexynylene, and the like.
The term “alkoxy” refers to linear or branched oxy-containing radicals each having alkyl portions of one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkoxy radicals are “lower alkoxy” radicals having one to about ten carbon atoms and more preferably having one to about eight carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.
The term “alkoxyalkyl” refers to alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.
The term “aryl”, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term “aryl” embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl.
The terms “heterocyclyl”, “heterocycle” “heterocyclic” or “heterocyclo” refer to saturated, partially unsaturated and unsaturated heteroatom-containing ring-shaped radicals, which can also be called “heterocyclyl”, “heterocycloalkenyl” and “heteroaryl” correspondingly, where the heteroatoms may be selected from nitrogen, sulfur and oxygen. Examples of saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g. pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. morpholinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. Heterocyclyl radicals may include a pentavalent nitrogen, such as in tetrazolium and pyridinium radicals. The term “heterocycle” also embraces radicals where heterocyclyl radicals are fused with aryl or cycloalkyl radicals. Examples of such fused bicyclic radicals include benzofuran, benzothiophene, and the like.
The term “heteroaryl” refers to unsaturated aromatic heterocyclyl radicals. Examples of heteroaryl radicals include unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.) tetrazolyl (e.g. 1H-tetrazolyl, 2H-tetrazolyl, etc.), etc.; unsaturated condensed heterocyclyl group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl, etc.), etc.; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc.; unsaturated 3 to 6-membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc.; unsaturated 3- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) etc.; unsaturated condensed heterocyclyl group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. benzoxazolyl, benzoxadiazolyl, etc.); unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.) etc.; unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.) and the like.
The term “heterocycloalkyl” refers to heterocyclo-substituted alkyl radicals. More preferred heterocycloalkyl radicals are “lower heterocycloalkyl” radicals having one to six carbon atoms in the heterocyclo radical.
The term “alkylthio” refers to radicals containing a linear or branched alkyl radical, of one to about ten carbon atoms attached to a divalent sulfur atom. Preferred alkylthio radicals have alkyl radicals of one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylthio radicals have alkyl radicals which are “lower alkylthio” radicals having one to about ten carbon atoms. Most preferred are alkylthio radicals having lower alkyl radicals of one to about eight carbon atoms. Examples of such lower alkylthio radicals include methylthio, ethylthio, propylthio, butylthio and hexylthio.
The terms “aralkyl” or “arylalkyl” refer to aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.
The term “aryloxy” refers to aryl radicals attached through an oxygen atom to other radicals.
The terms “aralkoxy” or “arylalkoxy” refer to aralkyl radicals attached through an oxygen atom to other radicals.
The term “aminoalkyl” refers to alkyl radicals substituted with amino radicals. Preferred aminoalkyl radicals have alkyl radicals having about one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred aminoalkyl radicals are “lower aminoalkyl” that have alkyl radicals having one to about ten carbon atoms. Most preferred are aminoalkyl radicals having lower alkyl radicals having one to eight carbon atoms. Examples of such radicals include aminomethyl, aminoethyl, and the like.
The term “alkylamino” denotes amino groups which are substituted with one or two alkyl radicals. Preferred alkylamino radicals have alkyl radicals having about one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylamino radicals are “lower alkylamino” that have alkyl radicals having one to about ten carbon atoms. Most preferred are alkylamino radicals having lower alkyl radicals having one to about eight carbon atoms. Suitable lower alkylamino may be monosubstituted N-alkylamino or disubstituted N,N-alkylamino, such as N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino or the like.
The term “substituted” refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted.
For simplicity, chemical moieties that are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, an “alkyl” moiety can be referred to a monovalent radical (e.g. CH 3 —CH 2 —), or in other instances, a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., —CH 2 —CH 2 —), which is equivalent to the term “alkylene.” Similarly, in circumstances in which divalent moieties are required and are stated as being “alkoxy”, “alkylamino”, “aryloxy”, “alkylthio”, “aryl”, “heteroaryl”, “heterocyclic”, “alkyl” “alkenyl”, “alkynyl”, “aliphatic”, or “cycloalkyl”, those skilled in the art will understand that the terms alkoxy”, “alkylamino”, “aryloxy”, “alkylthio”, “aryl”, “heteroaryl”, “heterocyclic”, “alkyl”, “alkenyl”, “alkynyl”, “aliphatic”, or “cycloalkyl” refer to the corresponding divalent moiety.
The terms “halogen” or “halo” as used herein, refers to an atom selected from fluorine, chlorine, bromine and iodine.
The term carbocyclic biaryl refers to fused bicyclic moieties, typically containing 4-20 carbon atoms. An example is naphthalene. The biaryl groups may contain 1-4 heteroatoms. Examples include indoles, isoindoles, quinolines, isoquinolines, benzofurans, isobenzofurans, benzothiophenes, benzo[c]thiophenes, benzimidazoles, purines, indazoles, benzoxazole, benzisoxazole, benzothiazole, quinoxalines, quinazolines, cinnolines, and the like.
The terms “compound” and “drug” as used herein all include pharmaceutically acceptable salts, co-crystals, solvates, hydrates, polymorphs, enantiomers, diastereoisomers, racemates and the like of the compounds and drugs having the formulas as set forth herein.
Substituents indicated as attached through variable points of attachments can be attached to any available position on the ring structure.
As used herein, the term “effective amount of the subject compounds,” with respect to the subject method of treatment, refers to an amount of the subject compound which, when delivered as part of desired dose regimen, brings about management of the disease or disorder to clinically acceptable standards.
“Treatment” or “treating” refers to an approach for obtaining beneficial or desired clinical results in a patient. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of symptoms, diminishment of extent of a disease, stabilization (i.e., not worsening) of a state of disease, preventing spread (i.e., metastasis) of disease, preventing occurrence or recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).
Pharmaceutical Compositions
The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients.
As used herein, the term “pharmaceutically acceptable carrier or excipient” means a non-toxic, inert solid, semi-solid, gel or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha-(α), beta-(β) and gamma-(γ) cyclodextrins; 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 as propylene 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.
The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
Liquid dosage forms for oral administration include 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, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene 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.
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 suspension or emulsion, such as INTRALIPID®, LIPOSYN® or OMEGAVEN®, or solution, in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. INTRALIPID® is an intravenous fat emulsion containing 10-30% soybean oil, 1-10% egg yolk phospholipids, 1-10% glycerin and water. LIPOSYN® is also an intravenous fat emulsion containing 2-15% safflower oil, 2-15% soybean oil, 0.5-5% egg phosphatides 1-10% glycerin and water. OMEGAVEN® is an emulsion for infusion containing about 5-25% fish oil, 0.5-10% egg phosphatides, 1-10% glycerin and water. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP 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.
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.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene 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.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one 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-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 polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
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 polyethylene 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.
Dosage forms for topical or transdermal administration of a compound of this invention 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, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches 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.
For pulmonary delivery, a therapeutic composition of the invention is formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system. Solid or liquid particulate forms of the active compound prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, particularly aerosolized antibiotics, is known in the art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No. 5,508,269 to Smith et al., and WO 98/43650 by Montgomery, all of which are incorporated herein by reference). A discussion of pulmonary delivery of antibiotics is also found in U.S. Pat. No. 6,014,969, incorporated herein by reference.
By a “therapeutically effective amount” of a compound of the invention is meant an amount of the compound which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
In certain embodiments, one or more therapeutic agents of the dosage unit may exist in an extended or control release formulation and additional therapeutic agents may not exist in extended release formulation. In one embodiment, the extended release contemplates the substantially continuous delivery of drug over an extended period of time, such as greater than one, two, three, four or more weeks. For example, an agent described herein may exist in a controlled release formulation or extended release formulation in the same dosage unit with another agent that may or may not be in either a controlled release or extended release formulation. Thus, in certain embodiments, it may be desirable to provide for the immediate release of one or more of the agents described herein, and the controlled release of one or more other agents. The rate of delivery may be altered, for example, by varying the lactide/glycolide ratio in a poly(D,L-lactide-co-glycolide) encapsulation. Other polymers that may be used include polyacetal polymers, polyorthoesters, polyesteramides, polycaprolactone and copolymers thereof, polycarbonates, polyhydroxybuterate and copolymers thereof, polymaleamides, copolyaxalates and polysaccharides. In one embodiment of the invention, a compound of formula I is formulated with an extended release component, such as a coated extended release matrix, an extended release matrix, or an extended release bead matrix. In one example, a compound of Formula I is used in combination with a polymer matrix (e.g., Eudragit), Hydroxypropyl methyl cellulose (HPMC) and/or a polymer coating (e.g., Eudragit). Such formulations can, for example, be compressed into solid tablets or granules or formed into pellets for capsules or tablets. Extended release oral formulation can be prepared using additional methods known in the art. For example, a suitable extended release form of a compound of Formula I may be a matrix tablet composition. Suitable matrix forming materials include, for example, waxes (e.g., carnauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols), oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow, palm dil, and soya bean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and polyethylene glycol). Other suitable matrix tabletting materials are microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, ethyl cellulose, with other carriers, and fillers. Tablets may also contain granulates, coated powders, or pellets. Tablets may also be multi-layered. Multi-layered tablets are especially preferred when the active ingredients have markedly different pharmacokinetic profiles. Extended release formulations can be made by spray drying polymer-drug mixtures, emulsion-based technologies, coacervation based technologies, film casting, extrusion based technologies and other processes to manufacture polymer-drug microparticles possessing an extended release profile. Examples of suitable extended release technologies that can be used to incorporate the compounds of formula I described herein include, without limitation, the MEDISORB® technology, as described in, for example, U.S. Pat. No. 6,264,987 to Wright, U.S. Pat. No. 5,654,008 and/or U.S. Pat. No. 5,792,477, for example; the PROLEASE® technology, as described, for example in U.S. Pat. No. 6,358,443 to Herbert; the technologies described by Southern Research Institute, as described for example in U.S. Pat. No. 6,306,425; and “Method of Preparing Sustained Release Microparticles,” U.S. Application No. 60/441,946, filed Jan. 23, 2003, and the technologies described by Alza Corp., including the ALZAMER® Depot injection technology. The contents of these patents are incorporated herein by reference in their entirety.
EXAMPLES
The compounds and processes of the present invention will be better understood in connection with the following examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.
Example 1
Synthesis of Compound 21A
































tert-butyl((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(((S)-1-((4-methoxybenzyl)oxy)-propan-2-yl)amino)-3-methylbutyl)(methyl)carbamate, EMF-2
This compound was synthesized according to literature procedure ( J. Am. Chem. Soc . Marcaurelle, L., et al, accepted).









tert-Butyl-((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(2-fluoro-N-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-nitrobenzamido)-3-methylbutyl)(methyl)carbamate, EMF-4
To a stirred solution of EMF-2 (21 g, 40.0 mmol, 1 equiv) and 2-fluoro-3-nitrobenzoyl chloride (20.36 g, 100 mmol, 2.5 equiv) in CH 2 Cl 2 (120 mL) at 0° C. was added triethyl amine (NEt 3 ) (27.7 mL, 200 mmol, 5 equiv). The reaction was allowed to warm to room temperature as it stirred, and no starting material remained after 1.5 h. H 2 O (50 mL) was added to the reaction and it was extracted with CH 2 Cl 2 (2×100 mL). The combined organic portion was dried over MgSO 4 , filtered, and concentrated. The crude material was purified by silica gel chromatography in Ethyl Acetate (EtOAc)/hexanes (10%→30%) to give the product. HRMS (ESI) calculated for C 35 H 55 FN 3 O 8 Si [M+H] + : 692.3737, found: 692.3764.









tert-Butyl(((2R,3R)-5-(S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-methyl-10-nitro-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, EMF-5
To a stirred solution of EMF-4 (24.5 g, 35.4 mmol, 1 equiv) in N,N-Dimethylformamide (DMF, 708 mL) was added CsF (10.76 g, 70.8 mmol (2 equiv). The resulting suspension was heated to 85° C. for 5 h. The solvent was then removed under reduced pressure, and the crude solid was dissolved in EtOAc (250 mL), washed with H 2 O (1×100 mL) and brine (1×100 mL), dried over Na 2 SO 4 , filtered, and concentrated. The product was used in the next reaction without purification. HRMS (ESI) calcd for C 29 H 39 N 3 NaO 8 [M+Na] + : 580.2629, found: 580.2614.









tert-Butyl(((2R,3R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, EMF-6
A mixture of compound EMF-5 (7.51 g, 13.47 mmol, 1 equiv) and Pd/C (1.43 g, 1.347 mmol, 0.1 equiv) in EtOH (500 mL) were stirred under H 2 at 40° C. No starting material remained after 2 h, and the reaction was cooled, filtered through Celite, and concentrated. The crude material was used in the next reaction without purification. The product (0.852 g, 1.614 mmol) was taken up in 10% aqueous NaHCO 3 (10 mL) in dioxane (60 mL), and a solution of FmocCl (2.088 g, 8.07 mmol, 5 equiv) in dioxane (5 mL) at 0° C. was added via syringe. The resulting cloudy solution was allowed to stir for 15 min at 0° C. and then room temperature overnight. The solution became clear as it warmed to room temperature. The reaction was quenched with sat. NH 4 Cl solution and extracted with EtOAc (2×100 mL). The combined organic portion was dried over MgSO 4 , filtered, and concentrated. The crude material was purified by silica gel chromatography (EtOAc/hexanes gradient). HRMS (ESI) calcd for C 44 H 51 N 3 NaO 8 : 772.3568 [M+Na] + found: 772.3593.









Allyl (((2R,3R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, EMF-7
To a stirred solution of EMF-6 in CH 2 Cl 2 (200 mL, 0.1 M) was added 2,6-lutidine (9.94 mL, 85 mmol, 4.0 equiv), and TBSOTf (14.7 mL, 64 mmol, 3.0 equiv). The mixture was stirred for 2 h and then quenched with saturated NH 4 Cl solution and extracted with EtOAc. The combined organic extracts were dried over MgSO 4 , filtered, and concentrated. The resulting oil was dissolved in CH 2 Cl 2 (120 mL) and cooled to −78° C. before triethylamine (12.5 mL, 90 mmol, 5.0 equiv) and allyl chloroformate (1.78 mL, 16.7 mmol, 1.0 equiv) were added. After 10 minutes, the reaction was quenched with saturated NH 4 Cl and extracted with CH 2 Cl 2 . The combined organic extracts were dried over MgSO 4 , filtered, and concentrated. The crude material was purified by silica gel chromatography (EtOAc/hexanes 30%→50%) to give the product (12.2 g, 78%).
The product (12.2 g, 16.6 mmol, 1.0 equiv) was then dissolved in CH 2 Cl 2 (200 mL) and pH 7 buffer (15 mL). The mixture was cooled to 0° C. and dichloro dicyano quinone (DDQ; 5.66 g, 24.9 mmol, 1.5 equiv) was added. The mixture was stirred for 10 minutes at 0° C. and then 1 h at room temperature before quenching with H 2 O. The product was then extracted with CH 2 Cl 2 , and the combined organic portion was washed with saturated NaHCO 3 before activated carbon was added. The mixture was then filtered through Celite, and the Celite was washed several times with hot CH 2 Cl 2 . The filtrate was concentrated and the crude material was purified by silica gel chromatography (0%→5% MeOH/CH 2 Cl 2 ) to give the product (10.0 g, 98%). HRMS (ESI) calcd for C 35 H 40 N 3 O 7 [M+H] + : 614.2861, found: 614.2840.
General Protocol A for Cleavage of the Compound from the Lantern and its Characterization for Qualitative Analysis
A portion of each product was used to assure that each reaction was complete. To complete this qualitative analysis and characterization, one segment of a lantern (¼) was severed and placed in a polypropylene vial (or 96-well plate) and HF/pyridine (200 μL) was added to completely submerge the lantern. The reaction was allowed to sit at room temperature for 3 h. The reaction was then quenched by slow addition of methoxytrimethylsilane (400 μL, >2 equiv). After 15 min, the quenched solution was then removed and combined with additional methanol (MeOH) washes (2×200 μL). The product was dried under reduced pressure to yield a solid which was dissolved in dimethyl sulfoxide (DMSO) and analyzed by ultraperformance liquid chromatography/mass spectrometer (UPLC/MS).









Allyl (((2R,3R)-10-amino-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate/EMF-31
Core 1B was synthesized as shown on the scheme 1 above. Core 1B (353 mg, 0.576 mmol) was rigorously dried by evaporating with benzene (3×5 mL) to azeotrope water. The white solid was dried under reduced pressure overnight. SynPhase L-series alkyl tethered diisopropylarylsilane lanterns (32 Lanterns, ˜15 μmol/lantern) were prepared for loading by washing with CH 2 Cl 2 (3×20 min) and dried overnight under reduced pressure. The lanterns were then activated in an oven-dried vial by addition of 3% TfOH in CH 2 Cl 2 . The vial was shaken for 10 minutes, and the lanterns turned bright red. The liquid was removed, 2,6-lutidine was added, and the lanterns were shaken until the red color disappeared. A small amount of CH 2 Cl 2 was added to the lanterns followed by core 1B (353 mg, 0.576 mmol, 1.2 equiv) in CH 2 Cl 2 . Enough CH 2 Cl 2 was added to cover the lanterns, and the reaction was shaken for 60 hours. The reaction solvent was then removed, and the lanterns were washed with CH 2 Cl 2 (2×8 mL) and DMF (2×8 mL). The lanterns were shaken in 20% piperidine in DMF (8 mL) for 30 minutes to remove the Fmoc protecting group. The lanterns were then washed with DMF (2×8 mL), 3:1 THF/H 2 O (1×8 mL), 3:1 THF:isopropanol (1×8 mL), THF (1×8 mL), and CH 2 Cl 2 (2×8 mL). General protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete. LRMS (M+H) + : 392.19.









Allyl (((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate/EMF-32
To EMF-31 (26¾ lanterns, 0.401 mmol) in CH 2 Cl 2 (8 mL) was added 1-isocyanatonaphthalene (1.357 g, 8.02 mmol, 20 equiv). The vial was sealed and shaken at room temperature for 36 h. The reaction mixture was then removed and the lanterns were washed with CH 2 Cl 2 (1×8 mL), DMF (2×8 mL), THF/H 2 O (3:1, 1×8 mL), THF/isopropanol (3:1, 1×8 mL), THF (1×8 mL), and CH 2 Cl 2 (2×8 mL). General protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete. LRMS (M+H) + : 561.20.









1-((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-2-((methylamino)methyl)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-10-yl)-3-(naphthalen-1-yl)urea/EMF-37
To EMF-32 (26½ lanterns, 0.383 mmol) in THF (8 mL) was added dimethylbarbituric acid (1.798 g, 11.51 mmol, 30 equiv) and Pd(PPh 3 ) 4 (444 mg, 0.384 mmol, 1.0 equiv). The vial was sealed and the reaction was allowed to shake at room temperature overnight. The reaction mixture was then removed, and the lanterns were washed with CH 2 Cl 2 (2×8 mL) and DMF (5×8 mL). The lanterns were then shaken in DMF (8 mL) overnight. The solvent was removed and the lanterns were washed with DMF (2×8 mL), THF/H 2 O (3:1, 1×8 mL), THF/isopropanol (3:1, 1×8 mL), THF (1×8 mL), and CH 2 Cl 2 (2×8 mL). General protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete. LRMS (M+H) + : 477.22.









N-(((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo-[b][1,4]dioxine-6-sulfonamide/Compound 21A: EC 50 =770 nM
To EMF-37 (11¼ lanterns, 0.169 mmol) in CH 2 Cl 2 (8 mL) was added 2,6-lutidine (490 uL, 4.22 mmol, 25 equiv) and 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride (792 mg, 3.38 mmol, 20 equiv). The reaction was allowed to shake for 60 h, and the reaction mixture was removed. The lanterns were washed with CH 2 Cl 2 (1×8 mL), DMF (2×8 mL), THF/H 2 O (3:1, 1×8 mL), THF/isopropanol (3:1, 1×8 mL), THF (1×8 mL), and CH 2 Cl 2 (2×8 mL). General Protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete. The remaining lanterns were then cleaved according to General Protocol A to give the product as a white solid. LRMS (M+H) + : 675.21.
Example 2
Synthesis of Compound-20A









N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-4-methoxy-N,3-dimethylbenzenesulfonamide/Compound 20A/EC 50 =0.314 uM
Compound-20A was synthesized in the same manner as Compound-21A above except p-methoxy-m-methyl sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.
Example 3
Synthesis of Compound-1A



















Compound No.
Stereochemistry




1A
2R, 3R, 5S


1B
2S, 3S, 5R


1C
2R, 3R, 5R


1D
2S, 3S, 5S


1E
2R, 3S, 5S


1F
2S, 3R, 5R


1G
2R, 3S, 5R


1H
2S, 3R, 5S























N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-4-methoxy-N-methylbenzenesulfonamide/Compound-1A/EC 50 =0.423 μM
Compound-1A was synthesized in the same manner as Compound-21A above except p-methoxy sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.
Example 4
Synthesis of Compound-2A



















Compound No.
Stereochemistry




2A
2R, 3R, 5S


2B
2R, 3R, 5R

















N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N,4-dimethylbenzenesulfonamide/Compound-2A/EC 50 =5.5 uM
Compound-2A was synthesized in the same manner as Compound-21A above except p-methyl sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.









N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(1-methyl-1H-indol-4-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N,4-dimethylbenzenesulfonamide/Compound-3A
Compound 3A was synthesized using a procedure similar to the synthesis of Compound-21A described above.









4-Chloro-N-(((2R,3R)-10-(3-(4-fluorophenyl)ureido)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methylbenzenesulfonamide
Compound-30A was synthesized in the same manner as Compound-21A above except that p-fluorobenzene isocyanate was used in place of 1-isocyanatonaphthalene in the reaction with the aniline and p-chloro sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.









N-(((2R,3R)-10-(3-(2,3-Dichlorophenyl)ureido)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-4-methoxy-N-methylbenzenesulfonamide
Compound-32A was synthesized in the same manner as Compound-21A above except that o,m-dichlorobenzene isocyanate was used in place of 1-isocyanatonaphthalene in the reaction with the aniline and p-methoxy sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.
The compounds were analyzed for activity utilizing methods described in Chou et al. (ACS Chem. Biol. 2010 Aug. 20; 5(8):729-34).
Cell Culture and Reagents:
INS-1E cells (generously provided by C. Wollheim and P. Maechler, University of Geneva) were maintained in RPMI 1640 containing 11 mM glucose, 10% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, cultivated at 37° C. with 5% CO 2 in a humidified atmosphere, and split every week. Recombinant rat IL-1β and recombinant mouse TNF-α were purchased from R&D Systems. Recombinant mouse IFN-γ, Griess reagent, and dexamethasone were purchased from Sigma. CellTiter-Glo and Caspase-Glo 3/7 reagents were purchased from Promega. Alsterpaullone and Ro 31-8220 were purchased from EMD Biosciences. The pyrazole derivatives were purchased from Maybridge. CHIR99021 was synthesized as described in Wang et al. (Proc. Natl. Acad. Sci. U.S.A. 106, 1427-1432, 2009).
High-Throughput Screening for Compounds Affecting Cellular ATP Levels:
INS-1E cells were seeded at 10,000 cells per well using a Multidrop Combi (Thermo Labsystems) in white optical 384-well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 μL of RPMI containing 1% FBS and a combination of cytokines (10 ng mL- 1 IL-1β, 50 ng mL −1 IFN-γ, 25 ngmL −1 TNF-α) was added to every well. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), 0.1 μL of each compound was added. After 48 h, medium was removed and 20 μL of CellTiter-Glo reagent was added. Luminescence was measured after 10 min of incubation using an EnVision plate reader (PerkinElmer).
Screening Data Analysis:
Instrument output files were processed using Pipeline Pilot (Accelrys) and input to MATLAB (The MathWorks) for data normalization. Compound performance scores relative to a distribution of mock-treated (DMSO) wells were calculated using a revised version of the scoring system underlying ChemBank. (Seiler et al., Nucleic Acids Res. 36, D351-359, 2008). The role of replicate treatments was further developed as follows: first, mock-treatment distributions were modeled using all mock-treated wells measured on a single day, regardless of their nominal replicate; second, per-compound scores weighted each in-plate background-subtracted measurement by the uncertainty in that measurement, using the method of maximum likelihood. The uncertainty in a single background-subtracted measurement was estimated using the number of mock-treated wells on the plate and, as a measure of the assay noise, the standard deviation of the per-day mock-treatment distribution. The signal, a weighted average of differences, was scaled by the noise, the standard deviation of the mock treatment distribution.
Measurement of Cellular Nitrite Production:
INS-1E cells were seeded and treated as described for high-throughput screening. After treatment with cytokine and compounds for 48 h, 10 μL of modified Griess reagent (1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 60% acetic acid) was added to each well. After 5 min of incubation at RT, the absorbance at 540 nm was measured using an EnVision (Perkin Elmer) plate reader.
Caspase-3 Activity Assay:
INS-1E cells were seeded at 5,000 cells per well in white optical 384-well plates and treated as described for highthroughput screening. After treatment with cytokines and compounds for 48 h, medium was removed and 20 μL Caspase-Glo 3/7 reagent was added. Luminescence was measured after 2 h of incubation using an EnVision plate reader. The data from the screening is tabulated in Table 3 below.
Glucose-Stimulated Insulin Secretion:
INS-1E cells were seeded in 96-well plates at 20,000 cells per well and incubated for 48 h in 100 μL of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. Cells were washed and incubated for 2 h in KRBH (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO 3 , 0.5 mM NaH 2 PO 4 , 0.5 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH 7.4, 0.1% BSA) without glucose. Cells were subsequently incubated with KRBH containing 2 or 15 mM glucose for 1 h. The supernatant was taken for measurement of released insulin, and 100 μL of acidified ethanol was added to each well for extraction and measurement of cellular insulin content. Insulin was measured with a rat insulin ELISA kit (Alpco).
The activity data for some of the compounds of the invention is tabulated in Table 3 below. In these assays, the following grading was used: I>0.1 μM; II>10 μM; III>20 μM; IV>25 μM.








TABLE 3




Compound No.
CTG EC 50 in HTS (μM)
Caspase EC 50 (μM)




1A
I
I


1B
IV
IV


1C
III
IV


1D
III
IV


1E
IV
IV


1F
III
IV


1G
IV
IV


1H
NT
NT


2A
II
IV


2B
III
III


3A
IV
II


4A
IV
IV


5A
I
IV


6A
IV
IV


7A
IV
IV


8A
IV
IV


9A
IV
IV


10A
III
IV


11A
II
IV


12A
IV
IV


13A
IV
IV


14A
IV
IV


15A
IV
IV


16A
II
IV


17A
IV
IV


18A
IV
IV


19A
IV
IV


20A
I
I


21A
I
II


22A
NA
NA


23A
IV
IV


24A
IV
IV


25A
IV
IV


26A
IV
IV


27A
IV
IV


28A
NT
NT


29A
NT
NT


30A
IV
IV


31A
III
III


32A
III
IV


33A
IV
IV


34A
IV
IV


35A
IV
IV


36A
IV
IV


37A
IV
IV


37B
IV



38A
IV
IV


39A
IV
IV


40A
IV
IV


41A
IV
IV


42A
IV
IV


43A
IV
IV


44A
IV
IV


45A
IV
IV


46A
IV
IV


47A
IV
IV


48A
IV
IV.



IA = Inactive

NA = Not Applicable

NT = Not Tested.










































































Measurement of Cellular ATP Levels.
INS-1E cells were seeded at 10,000 cells/well using a Multidrop Combi (Thermo Labsystems) in white optical 384-well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 μL RPMI containing the treated compound, 1% FBS and a combination of cytokines (10 ng/mL IL-1β, 50 ng/mL IFN-γ, 25 ng/mL TNF-α) was added to every well. After incubation for 48 hr, medium was removed and 20 μL CellTiter-Glo reagent was added. Luminescence was measured after 10-min incubation using an EnVision plate reader (PerkinElmer).
Gene-Expression Profiling and Gene-Set Enrichment Analysis (GSEA).
We performed gene-expression profiling on INS-1E cells using the Affymetrix Rat 230 2.0 array. Data normalization and gene-set enrichment analysis (GSEA) were performed using GenePattern (http://genepattern.broadinstitute.org) as described in Subramanian A et al. (2005) Proc. Natl. Acad. Sci. USA 102:15545-15550.
GAS Reporter Assay.
INS-1E cells were transfected with plasmids (GAS reporter assay kit) using DharmaFECT reagent according to the manufacturer's protocol (SABiosciences) in white optical 384-well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 μL RPMI containing the treated compound, 1% FBS and a combination of cytokines (10 ng/mL IL-1β, 50 ng/mL IFN-γ, 25 ng/mL TNF-α) was added to every well. After treatment with cytokines and compounds for 18 hr, luminescence was measure using Dual-Glo according to the manufacturer's protocol (Promega).
RNA Interference and Western Blotting.
siRNAs (100 nM) were transfected into INS-1E cells (5,000 cells/well in a 384-well plate) using DharmaFECT reagent. Transfected cells were cultured for 72 hr, then collected for Western blot analysis and cell-based assays. For Western blotting, cells were lysed in RIPA buffer. Total protein was separated by 4-12% SDS-PAGE and transferred to a PVDF membrane. Blots were developed using the chemiluminescence detection system SuperSignal (Thermo Fisher Scientific) and light emission was captured using an Imaging Station 4000MM (Carestream).
Quantitative PCR for mRNA Levels.
Following knock-down by siRNA, cells were lysed and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA was reverse transcribed with random primers using the High Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystems). Quantitative PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR machine using primers for rat Mcm6 and Blvrb from SABiociences.
Kinase Profiling.
Kinase profiling was performed by Millipore (KinaseProfiler) according to the manufacturer's protocols. ATP concentrations were within 15 μM of the apparent KM for each enzyme.
SILAC and Mass Spectrometry.
We performed stable isotope labeling of amino acids in cell culture (SILAC) on INS-1E cells, followed by mass spectrometry to identify proteins enriched in the absence of soluble BRD0476 competitor, as described in Ong S E et al. (2009) Proc. Natl. Acad. Sci. USA 106:4617-4622.
Purification of Recombinant USP9X.
For each purification, fifty 25-cm dishes of confluent HEK-293 cell lines stably expressing wild-type USP-9X (provided by Dr. Dario Alessi, University of Dundee, Scotland) were employed. Cells were washed twice with ice-cold PBS and lysed in 1 ml of ice-cold lysis buffer. The combined lysates were centrifuged at 26000 g for 30 min at 4° C. and the supernatant incubated with 0.2 ml of rabbit IgG-agarose beads (Sigma) for 1 h at 4° C. The IgG-agarose was washed extensively with lysis buffer containing 0.15 M NaCl, then with several washes in buffer B prior to incubation with 0.250 ml of buffer B containing 0.1 mg of TEV protease (Invitrogen). After 3 h at 4° C.˜70-90% of the TAP-tagged protein had been cleaved from the IgG-agarose and the eluted protein was incubated with 0.1 ml of rabbit calmodulin-Sepharose (Roche) equilibrated in buffer C. After 1 h at 4° C., the calmodulin-Sepharose was washed with buffer C. To elute the protein, the calmodulin-Sepharose was then incubated with 0.1 ml of buffer D for 10 min at 4° C. The eluate was removed from the beads and the elution repeated two or three times. To remove the NaCl present in the buffer containing the eluate protein, the eluates were centrifuged at 1500 g for 1 min at 4° C. in protein desalting spin columns. Lysis buffer contained 50 mM Tris/HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% (w/v) Nonidet P40, 1 mM sodium orthovanadate, 10 mM sodium b-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM DTT (dithiothreitol) and complete proteinase inhibitor cocktail (one tablet/50 ml). Buffer B contained 50 mM Tris/HCl (pH 7.5), 0.15 M NaCl, 0.27 M sucrose, 1% (w/v) Nonidet P40 and 1 mM DTT. Buffer C contained 50 mM Tris/HCl (pH 7.5), 0.15 M NaCl, 1 mM MgCl 2 , 1 mM imidazole, 2 mM CaCl 2 , 0.27 M sucrose and 1 mM DTT. Buffer D contained 50 mM Tris/HCl (pH 7.5), 20 mM EGTA, 150 mM NaCl and 5 mM DTT. TBS-Tween buffer contained 50 mM Tris/HCl (pH 7.5), 0.15 M NaCl and 0.2% (v/v) Tween 20.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.