NZ627559B2 - Inhibitors of notch signalling pathway and use thereof in treatment of cancers - Google Patents

Inhibitors of notch signalling pathway and use thereof in treatment of cancers Download PDF

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Publication number
NZ627559B2
NZ627559B2 NZ627559A NZ62755912A NZ627559B2 NZ 627559 B2 NZ627559 B2 NZ 627559B2 NZ 627559 A NZ627559 A NZ 627559A NZ 62755912 A NZ62755912 A NZ 62755912A NZ 627559 B2 NZ627559 B2 NZ 627559B2
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New Zealand
Prior art keywords
formula
tert
pyridinamine
butylphenoxy
notch
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NZ627559A
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NZ627559A (en
Inventor
Rajwinder Lehal
Freddy Radtke
Viktoria Reinmuller
Jieping Zhu
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Ecole polytechnique fédérale de Lausanne (EPFL)
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Priority claimed from EP11010130.0A external-priority patent/EP2606884A1/en
Application filed by Ecole polytechnique fédérale de Lausanne (EPFL) filed Critical Ecole polytechnique fédérale de Lausanne (EPFL)
Publication of NZ627559A publication Critical patent/NZ627559A/en
Publication of NZ627559B2 publication Critical patent/NZ627559B2/en

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    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
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Abstract

The present disclosure relates to compounds of formulae II, III and IV where the substituents are as defined herein as inhibitors of Notch signalling pathway in treating and/or preventing cancers, wherein the Notch dependent cancer is selected from the group comprising T cell-Acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL), breast cancer, pancreatic cancer, prostate cancer, melanoma, brain tumors, tumor angiogenesis, and colorectal cancer. An example is the compound is: 6-(4-tert-butylphenoxy)pyridin-3-amine. kemia (T-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL), breast cancer, pancreatic cancer, prostate cancer, melanoma, brain tumors, tumor angiogenesis, and colorectal cancer. An example is the compound is: 6-(4-tert-butylphenoxy)pyridin-3-amine.

Description

Inhibitors of Notch signalling pathway and use thereof in ent of cancers FIELD OF THE INVENTION The present invention relates to the use Of inhibitors OfNotch signalling y in particular 6-(4-Tert-Buty1phen0xy)PyridinAmine (13) (CAS number 2184571) and its derivatives, in the treatment and/or prevention Of cancers.
OUND OF THE INVENTION The Notch signalling pathway represents a al component in the molecular ts that control cell fate during development, cell survival and cell proliferation (Shih IeM, Wang TL in Cancer Res 2007;67(5):1879—82). Aberrant activation of this pathway contributes to tumorigenesis. The Notch family members are being revealed as oncogenes in an ever- increasing number Of cancers. The role OfNotch in human cancer has been highlighted recently by the ce Of activating mutations and amplification OfNotch genes in human cancer and by the tration that genes in the Notch signalling y could be potential therapeutic targets. It has become clear that one Of the major therapeutic targets in the Notch y are the Notch receptors, in which y—secretase inhibitors prevent the generation Of the oncogenic (intracellular) domain of Notch molecules and suppress the Notch ty.
Though significant progress has been made in dissecting the complex workings Of this signalling pathway, there are very limited Options available for Notch inhibitors. However, the pioneering class OfNotch tors is already in clinical trials for few cancer types, such as y- secretase inhibitors MK0752 of Merck Sharp & Dohme Corp. MK0752, and RO4929097 (Roche), a synthetic small molecule, inhibits the Notch signalling pathway, which may result in ion Of growth arrest and apoptosis in tumor cells in which the Notch signalling pathway is overactivated.
One of the drawbacks of use of y-secretase inhibitors to block Notch signaling, as currently on the market or uner investigation, is their wide range Of additional targets such as amyloid precursor protein as well as non— selectivity in blocking Notch signalling via all four ligands (Notchl , 2,3 and 4). Due to their ability to block Notch signalling via all four receptors y- secretase tors are known to cause goblet cell metaplasia in the intestine. In addition, some of the hematological malignancies and solid tumors harbor mutations in the Notch receptors (such as chromosomal translocations) resulting in constitutive expression of dominant active form ofNICD independent of cleavage by y-secretase x. ore these tumors fail to d to y—secretase inhibitors treatment.
Therefore, there is still a need to identify and develop further specific and selective inhibitors ofNotch signalling y useful for treating and/or preventing cancers.
SUMMARY OF THE INVENTION The present invention concerns an 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) of Formula I 0 N Formula I or one of its derivatives having Notch signalling pathway inhibition properties, salts, solvates, tautomers, isomers thereof for use in the treatment and/or prevention of a cancer.
A r object of the present ion is to provide a pharmaceutical composition comprising pharmaceutical composition comprising 6-(4-Tert-Butylphenoxy)Pyridin—3- Amine (I3) of Formula I, or one of its tives having Notch signalling pathway inhibition properties, or pharmaceutically acceptable salts, solvates, tautomers, isomers thereof, and a ceutically acceptable carrier.
The invention also contemplates a kit comprising one or more doses of 6-(4-Tert- Butylphenoxy)PyridinAmine (I3), or one of its derivatives having Notch signalling pathway inhibition properties, for use in a method for treatment and/or prevention of cancer, optionally with reagents and/or instructions for use. 3 2012/057622 A further object of the invention is to provide the use of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) of Formula I or one of its derivatives having Notch signalling pathway inhibition ties, for inhibiting in vitro or in vitro the Notch signalling pathway in cells.
Another object of the invention is to provide a method of treating a subject for Notch dependent cancer.
DESCRIPTION OF THE FIGURES Figure 1 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (13) (CAS number 2184571) blocks NICD mediated Notch signalling tion. A) Nl-HeLa cells were co-transfected with pcDNA3.Notchl expression plasmid, pGL4.26-l2XCSL luciferase and SV40 renilla plasmids. DL4- and Nl-HeLa cells were cocultured in a 96 well plate in 1:1 ratio (20,000: 20,000 cells/well) and treated with DMSO or with 2, 5 and 10 uM of 13 and DAPT for 24 hours. The Notch pathway activation was measured by quantifying Notch signalling driven luciferase reporter assay. Treatment of DL4:Nl coculture assay with 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) and DAPT causes a concentration dependent decrease in Notch signalling activation. B) HeLa cells were transfected with NICD and treated with DMSO or with 2, 5, 10, 20 and 40 uM of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13). As a control co-cultured cells were also treated with 5, 10, 20 and 40 uM of DAPT. The pathway activation was measured using Notch driven luciferase reporter assay. 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treatment ofNICD expressing cells led to an attenuation of the signalling while DAPT ent had no effect on Notch signalling activation mediated by NICD. C) DL4:Nl and DL4:N2 coculture assay was treated with 13 and DAPT (each 10 uM) for 24 hours. The effect of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT on DL4-Nl and DL4-N2 driven y tion was ed by Notch driven luciferase activity. Both 13 and DAPT treatment block Notchl and Notch2 induced pathway activation. D) 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) inhibits pathway tion via intracellular domains ofNotchl (NICD) and Notch2 (N2-ICD).
Figure 2 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) ed inhibition h signalling can be rescued with increasing tration of MAMLl. HeLa cells were co- transfected 800ng ofNICD + 3 ug ofpCDNA3.l or 800ng ofNICD + 1 ug of MAMLl-FLAG or 800ng ofNICD + 3ug ofMAMLl-FLAG expression vectors. To measure Notch pathway activation, pGL4.26-l2xCSL luciferase plasmid was also introduced into the cells. SV40 renilla was used as an internal control. Cells ected with ent combinations and amounts of plasmid were treated with DMSO or increasing concentration of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) (l, 2.5, 5 and 10 uM) for 24 hours. l2xCSL driven luciferase activity was measured using dual luciferase assay system. In the absence of MAMLl, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) could block Notch signalling activations, but Notch inhibitory effect of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) was diminished with increasing amount of MAMLl.
Figure 3 shows ert-Butylphenoxy)PyridinAmine (I3) inhibits Notch signalling and downregulates its target genes in human cancer cell lines. A) RPMI 8402 cells were treated with DMSO, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT (10 uM) for 24 hours and ed for the expression ofNotch target genes, Hesl, cMyc and Dtxl by qRT-PCR.
Data normalized to HPRT as a house-keeping gene. B) Whole cell lysate from 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treated cells was analyzed by Western blot. Using dies against NICD (Vall744), Hesl and cMyc, the protein levels ofNICD and Notch target genes were determined. C and D) The human T-ALL cell lines HPB ALL and KOPTKl were treated with DMSO or 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) for 24 hours. Western blot es were performed using NICD (Vall744) and Hesl ic antibodes. Tubulin served as a loading control. E) Whole cell lysate from DMSO, 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treated PANCl cells (pancreatic cancer cell line) were analyzed by Western blot. Hesl protein levels were determined using Hesl specific antibodies. Statistical analyses were done using student’s two-tailed t.test. * = p value < 0.05.
Figure 4 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) induces a proliferative block in human cancer cells. Human T-ALL cell lines RPMI 8402 and KOPTKl, and pancreatic cancer cell line PANCl as well as nRas driven melanoma cells were seeded in a 96 well plate and d with 10 uM concentration of 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) and DAPT for several days. Their growth inhibitory effects were ed with cells treated with equal amount of DMSO. Using Alamar blue assay, the growth cs of RPMI 8402 and KOPTKl were followed for upto 6 days, while PANCl and nRas melanoma cells were monitored for 4 days. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment of RPMI 8402, KOPTKl. PANCl and nRas melanoma cells caused a significant reduction in their growth potential. Statistical analyses were done using student’s t.test. * = p value < 0.05. ns= not significant.
Figure 5 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (13) blocks NICD dependent growth of human cancer cells. A) DND41-Parental and DND41-NICD cells were treated with 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) and DAPT for 24 hours. n blot analyses were carried out for Hesl protein using Hesl specific dies. Both DAPT and 6-(4-Tert- Butylphenoxy)PyridinAmine (13) caused a downregulation of Hesl in DND41-Parental cells. DND4l-NICD cells showed a downregulation of Hesl only when treated with 6-(4- utylphenoxy)PyridinAmine (13). B) Five thousand Parental cells were seeded and treated with DMSO, 6-(4-Tert—Butylphenoxy)PyridinAmine (I3) and DAPT in a 96 well plate. Growth kinetics of the parental cell line was followed over 5 days using Alamar blue readout. Treatment of DND41-Parental cell line with both 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) and DAPT caused a proliferation arrest. C) rly, NICD cells treated with DMSO, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT and their growth kinetics were monitored using Alamar blue readout over 5 days. The treatment of DND41-NICD cells with DAPT did not have a significant impact on their proliferation, while 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment induced a proliferation arrest. D) Human breast cancer cell line HCC1187 harbors a SEC22B-Notch2 chromosomal translocation, thus leading to an expression of constitutively active form of NICD independent of cleavage by the y—secretase complex. This on renders this cell line insensitive to y—secretase tor treatment. E) Two thousand HCC1187 cells were seeded per well in a 96 well plate. The cells were treated with DMSO, y-secretase inhibitor DAPT and 13 for 6 days. Alamar blue readout was taken at day 0, day 2, day 4 and day 6.
Eight ates were used for each treatment and time point. The ent of HCCl 187 human breast cancer cell line with y-secretase inhibitor DAPT did not alter the growth kinetics when compared to DMSO treated counterparts, while 13 treatment caused statistically significant inhibition of cell proliferation. P values were calculated using Student’s t.test. * = p value < 0.05. ns= not significant.
Figure 6 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) s G0/Gl cell cycle arrest and apoptosis in human T cell acute lymphoblastic leukemia cell lines and human breast cancer cell line HCC1187. A) Human leukemic cell lines (RPMI8402, CUTLl, KOPTKl TALLl and HPBALL) were treated with 13 (10 uM). Percentage of Annexin V positive (apoptotic) cell population was measured using flow cytometry. B) Cell cycle analyses: RPMI8402, KOPTKl and TALLl cell lines were treated with 13 (10 uM) and stained with Ki67 and Hoechst stain to ine cell cycle status. The cell cycle analyses suggest that 13 treatment causes 20-30 % increase in cells arrested in GO/Gl phase of the cell cycle. C) HCC1187 cells were treated with DMSO or 10 uM of 13 and percentage of apoptotic population was measured using Annexin V stain. D) 13 treated HCC1187 cells analyzed for cell cycle status. Ki67 and Hoechst stain revealed that 13 induces GO/Gl arrest in HCC1187 cells.
Figure 7 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mimics genetic loss of Notch2 signalling phenotype in the . Loss ofNotch ling in the spleen leads to a ion in Marginal Zone B cells (MZB) cells in the spleen. A) Schematics of the mental plan. B) Mice (n=2) were treated with oil or 25 mg/kg of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) for 7 consecutive days. Spleens were analyzed on day 8.
Using B220 specific antibodies, B cells in the spleen were identified. MZB cells within the B cell compartment were detected using antibodies t CD23 and CD21 cell surface markers. The treatment of mice with 6-(4-Tert-Butylphenoxy)PyridinAmine (13) causes a significant reduction in the tage ofMZB cells in the spleen. B) 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treatment causes a reduction in the absolute numbers of MZB cells in the spleen when compared to vehicle treated animals.
Figure 8 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment increases latency of ia development in mice. A) NOD/SCID yc'/' mice were injected with 1 x 106 HPB ALL (luciferase expressing) cells. On day 15, leukemic cells were established in the bone marrow. Mice were treated with oil or 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) for every day. Mice were imaged on day 27 using Caliper IVIS (Xenogen) live g system.
Red and blue colour indicates the ity of luciferase signal and correlates with the number of leukemic cells. B) 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment blocks RPMI 8402 leukemic cell growth in xenotransplantation assay. NOD/SCID yc'/' mice were transplanted with 5 x 105 RPMI 8402 erase sing) cells. Leukemia development was followed using Caliper IVIS (Xenogen) live imaging system. On day 13, a daily treatment was started using oil (n=3) or 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) (n=4).
Animals were treated for 27 days (end point of the experiment).
Figure 9 shows 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment blocks MMTV- ErbB2 mouse mammary tumors. A) Hesl protein expression levels in MMTV-ErbB2 mammary tumors and normal age matched mammary glands (M.G) were compared using Anti-Hesl antibody. Western blot analyses showed a very high expression of Hesl protein in rbB2 y tumors. n served as a loading control. B) A single cell suspension of MMTV-ErbB2 mammary tumor was prepared and l x 106 cells were injected into the cleared fat pad of recipient FVB mice. Tumor formation was monitored on a regular basis. Once the tumor developed to a volume of 100-300 mm3’ mice were treated with oil (n=2) or 25 mg/kg of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) (n=2). Tumor volume was ed every 6-7 days. Mice treated with 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) exhibited a slow tumor progression compared to oil treated mice.
Figure 10 shows Notch inhibitory activity of chemical tives of 6-(4-Tert- Butylphenoxy)PyridinAmine (13). Different al derivatives of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) were tested in DL4-N1 coculture assay and Notch activity levels were measured using Notch driven rase reporter gene. Derivatives 13 -A, I3-B, I3-C, I3-E, I3-G, I3-H, I3-M and I3-N exhibit anti-Notch activity comparable to 6-(4- Tert-Butylphenoxy)PyridinAmine (13), while derivatives I3-F and 13-1 appear to have enhanced activity.
DETAILED DESCRIPTION OF THE INVENTION Although methods and als similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and als are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their ty. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In on, the materials, methods, and examples are illustrative only and are not intended to be limiting.
In the case of conflict, the present specif1cation, including def1nitions, will control. Unless def1ned otherwise, all technical and scientific terms used herein have the same meaning as is ly tood by one of skill in art to which the subject matter herein belongs. As used herein, the ing definitions are supplied in order to facilitate the understanding of the present invention.
As used herein, the term “comprise/comprising” is lly used in the sense of include/including, that is to say permitting the presence of one or more features or components. The terms “comprise” and “comprising” also ass the more restricted ones “consist” and “consisting”.
As used in the specification and claims, the singular form “ 3, (C a an” and “the” include plural nces unless the context clearly dictates otherwise.
For the ease ofreading, the term “compound(s) of the invention” or “compound(s) according to the invention" used throughout the description refers to the compound 6-(4-Tert- henoxy)PyridinAmine (13) (CAS number 2184571), derivatives of said 13, salts or solvates of the compound 13 or of the derivatives, and to isomers, including enantiomers, stereoisomers, rotamers, ers and racemates of the compound 13, chemical ed 13 compounds and derivatives of said 13 compounds.
As used herein the terms “subject” is well-recognized in the art, and, refers to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a t in need of treatment or a subject with a disease or disorder, such as cancer. However, in other embodiments, the subject can be a normal subject or a subject who has already undergone a treatment against cancer.
The term does not denote a particular age or sex. Thus, adult, children and newborn subjects, whether male or female, are intended to be covered.
The terms “cancer”, “cancer cells”, “cell proliferative diseases” and “cell erative disorders” as used herein refer to or describe the physiological condition in mammals that is lly characterized by unregulated cell . According to the present invention, cancer refers ably to solid tumors, such as brain, breast, prostate, colorectum, kidney, lung, a, or melanoma and liquid tumors, affecting the blood, such as leukemia. More preferably according to the present invention, cancers are Notch dependent cancers selected from the group comprising T cute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Mantle cell lymphoma, breast cancer, pancreatic cancer, te cancer, melanoma, brain tumors, tumor angiogenesis, colorectal cancer. Alternatively, the Notch dependent cancer is resistant to y—secretase inhibitor treatment. Examples of y—secretase inhibitor treatment comprise 1) Gamma secretase inhibitor RO4929097 and Cediranib Maleate in treating patients with advanced solid tumors (NCT01 13 1234), 2) Gamma-Secretase Inhibitor RO4929097 in Treating Young Patients With Relapsed or Refractory Solid Tumors, CNS Tumors, Lymphoma, or T-Cell Leukemia (NCT01088763), 3) Study of 2 in combination with Tamoxifen or Letrozole to treat early stage breast cancer 756717), 4) GDC-0449 and RO4929097 in ng ts with Advances or metastatic sarcoma (NCT01 154452) 5) 097 and Erlotinib Hydrochloride in treating patients with stage IV or ent Non-Small Cell Lung Cancer (NCT01193881), 6) Bicalutamide and RO4929097 in treating patients with previously treated prostate cancer (NCT01200810), 7) RO4929097 in treating patients with recurrent invasive s (NCT01269411), 8) A Notch signaling pathway inhibitor for patients with T-cell Acute Lymphoblastic Leukemia/Lymphoma (ALL) (NCT00100152) and 9) RO4929097 in treating patients with metastatic colorectal cancer (NCTOl 1 .
The Notch signalling pathway is evolutionarily ved and the basic molecular players in this pathway are ligands (Delta and Jagged), Notch receptors, and the transcription factors (Shih IeM, Wang TL in Cancer Res 2007;67(5):1879—82). Notch is a transmembrane heterodimeric receptor and there are four distinct members (Notch1, Notch2, Notch3 and ) in humans and rodents. In a physiologic condition, binding of the Notch ligand to its receptor initiates Notch signalling by releasing the intracellular domain of the Notch or (Notch-1CD) through a cascade of proteolytic cleavages by both u-secretase (also called tumor necrosis factor-(x—converting enzyme) and y—secretase. The released intracellular Notch-ICD then translocates into the s where it tes gene expression primarily by binding to a ubiquitous transcription factor, CBFl, suppressor of hairless, Lag-1 (C SL). This binding recruits transcription tors to the CSL complex and converts it from a transcriptional repressor into an activator, which turns on several downstream effectors. The physiologic functions h signalling are multifaceted, including maintenance of stem cells, specification of cell fate, and regulation of differentiation in development as well as in oncogenesis.
In cancers, molecular genetic alterations, such as somal translocation, point mutations, and chromosomal amplification at the Notch receptor loci, are the known mechanisms for W0 2013/093885 10 constitutive activation ofNotch pathway. Despite the different mechanisms, they all result in increased levels of intracellular Notch-IC. The nic potential ofNotch was first discovered in human T-cell acute lymphoblastic leukemia (T-ALL). While Notchl signalling is essential for normal development of T-cell progenitors, tutive activation ofNotchl signalling due to molecular genetic alterations is associated with T-ALL. For example, interstitial deletions of the extracellular portion of human Notchl due to (7;9) chromosomal translocation are associated with ~ 1% of T-ALL cases and activating point ons of Notchl are t in about 50% of T-ALL cases. Formation of T-cell leukemia/lymphoma was observed in a Notch-1CD transgenic mouse model, which indicates a causal role ofNotch activation in T-ALL development. In non—small cell lung cancer, chromosomal translocation (15;l9) has been identified in a subset of tumors, and the translocation is thought to e Notch3 transcription in tumors. In ovarian cancer, Notch3 gene amplification was found to occur in about 19% of tumors, and overexpression of Notch3 was found in more than half of the ovarian serous carcinomas. Similarly, Notch signalling activation has been shown in the pment of breast cancer. In animal models, constitutively active Notch4 expression causes mammary tumors in mice and Notchl-activating mutations contribute to the development of T-ALL. A recent study further shows that overexpression of activated Notchl and Notch3 in transgenic mice blocks mammary gland development and induces mouse breast . Notch signalling activation has also been ated in lung and bone asis of breast cancer cells. Overexpression ofNotch3 is sufficient to induce choroid plexus tumor formation in a mouse model, suggesting a role ofNotch3 in the development of certain types of brain tumors.
With the aim of conducting a High-Through put Screening (HTS) to identify novel modulators (inhibitors) ofNotch signalling, Applicants have established a coculture assay to induce a ligand-receptor mediated activation of the pathway. The coculture assay was established using Notch ligand DL4 and Notchl or specifically, because DL4-N1 ligand-receptor mediated pathway tion plays an important role in pathophysiological conditions such as tumor angiogenesis and the role ofNotchl receptor in inducing T cell leukemia. Since this assay depends on the expression and interaction between DL4 ligand and Notchl receptor, it provides an opportunity to interrogate ligand-receptor ctions-induced Notch signalling in a controlled manner. The urization of this assay into a 96 well plate and 384 well plate format helped Applicants to adapt this assay to conduct HTS. The use of this coculture assay to screen siRNA or small le libraries can lead to the identification W0 2013/093885 11 of proteins or chemical compounds that are able to modulate Notch signalling at different steps along the y. For example, a HTS using siRNA or small le libraries can yield modulators of the pathway able to act in the signal sending or signal receiving cells.
Small molecule or protein mediated alterations in the recycling or trafficking of the ligands and receptors to the plasma membrane can potentially block the Notch pathway and could be studied using this assay. In addition, this assay can also help identify proteins or chemical entities able to block ligand-receptor interactions, ADAMlO/l7 mediated SZ cleavage or y- secretase catalyzed S3 cleavage of the Notch or, nuclear translocation of the active form ofNotch or entities able to block transcriptional activation complex.
Applicants have also been able to screen three different chemical nd libraries (Microsource NIMDS, Prestwick and Maybridge Hit finder) that have led to the identification of several als, which are able to block Notch signalling at different levels along the pathway.
The use of the Notch-independent renilla system as an internal control allowed Applicants to eliminate cytotoxic chemical nds, thereby limiting the rate of false positive hits. In addition, this cell-based assay also helped to circumvent issues related to the cell permeability of the al compounds for filrther hit tion.
The development of DL4:Nl coculture assay system laid the tion for a HTS campaign.
This assay provided a robust and sensitive readout system to identify novel modulators (inhibitors) of the Notch pathway.
Applicants identified several chemical compounds for their ability to block the Notch pathway activation. Among those, they identified the compound 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) (CAS number 2184571) for its ability to block the Notch pathway activation.
Thus, the present ion relates to ert-Butylphenoxy)PyridinAmine (I3) of Formula I W0 2013/093885 12 Q N for use in the treatment and/or prevention of a cancer.
The t invention also encompasses chemical modifications of the 6-(4-Tert- Butylphenoxy)PyridinAmine (13) (CAS number: 2184571) to prolong their circulating lifetimes. Non-limiting examples of methods for transiently, or reversibly, ting drugs, including polypeptide-based drugs, are provided in US. Pat. Nos. 465 (issued in Jun. 19, 1990) and 6,342,244 (issued Jan. 29, 2002); and in US. published applications number US2006/0074024. One skilled in the art would typically find more details about PEG-based reagents in, for example, published applications WO2005047366, US2005 171328, and those listed on the NEKTAR PEG Reagent g® 2005-2006 (Nektar Therapeutics, San Carlos, Calif).
The present invention fiarther asses chemical derivatives of said 13 having Notch signalling pathway inhibition properties. Applicants have shown that 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) and its derivatives target Notch signalling at the transcriptional activation complex in the nucleus, human tumors resistant to etase tors due to above mentioned mutations are expected to respond to 6-(4-Tert- henoxy)PyridinAmine (13) treatment. In addition, 6-(4-Tert-Butylphenoxy)Pyridin- 3-Amine (13) appears to selectively target Notch signalling thus limiting its off-target toxic effects.
These derivatives all share the follow common structure: OX1) Preferably, in said derivatives X is O and position 3 (or para) is NH2.
W0 2013/093885 13 Most preferably, the derivative having Notch signalling pathway inhibition properties is selected from the non-limiting group comprising 122%R1 X 4 Formula 11 wherein m is an integer ed from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl—, Br- or 1-; R1, R2, R3, R4 are each independently selected from the group ting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, yl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 l, alkynyl; the subscript n is an integer independently ed from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8, or NHSOZR9; where R5, R6, R7, R8, R9 are each independently selected from the group consisting in selected from the group consisting in H, phenyl, 2- ,3- or 4- tuted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, pyl, tertButyl, or CH3 the subscript n is an integer independently selected from 1 to 15; Y is N or CH; Z is H, N02, OH, NRlORl l where R10 and R11 are each independently selected from the group consisting in H and (CH2)nCH3, NHCOR12 where R12 is selected from the group consisting of (CH2)nCH3, aromatic and heteroaromatics such as phenyl, naphthyl, pyrrolyl, fiaranyl, thiofiaranyl, pyrimidinyl, imidazolyl, COOR13 where R13 is selected from the group consisting of H, (CH2)nCH3, aromatic and heteroaromatics such as phenyl, naphthyl, pyrrolyl, fiaranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOle4 where R14 is selected from the group consisting of phenyl, 2- ,3- or 4, substituted phenyl, naphthyl, heteroaromatics such as pyrrolyl, furanyl, ranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer ndently selected from 1 to 15; <W>m R1\5: §:/@X Y Formula 111 W0 2013/093885 14 wherein m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is ed from F-, Cl—, Br- or 1-; R4, R15 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or thyl, pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, olyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, olyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group ting in , 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected from the group comprising pyrrolyl, filranyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; Y is N or CH; Z is H, N02, OH, l Where R10 and R11 are each independently selected from the group consisting in H, (CH2)nCH3, 2 Where R12 is (CH2)nCH3, aromatic and heteroaromatics ed from the group comprising phenyl, naphthyl, yl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, COORl3 Where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, olyl, NHSOle4 Where R14 is phenyl, 2- ,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from the group comprising pyrrolyl, filranyl, thiofuranyl, dinyl, imidazolyl, benzyl, (CH2)nCH3, the subscript n is an integer independently selected from 1 to 15; @@Qm X Formula IV wherein m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl—, Br- or 1-; R4 is H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, pyrrolyl, filranyl, thiofuranyl, dinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, l; the subscript n is an integer independently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, yl, filranyl, ranyl, dinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group consisting in phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected from the group comprising pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; Y is N or CH; Z is H, N02, OH, NR10R11 Where R10 and R11 are each ndently selected from the group consisting in H, (CH2)nCH3, NHCOR12 Where R12 is CH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, pyrrolyl, fiaranyl, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 Where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, pyrrolyl, fiaranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOzR14 Where R14 is phenyl, 2- ,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from the group comprising pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, CH3, the subscript n is an integer independently selected from 1 to 15; wherein m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl—, Br- or I-; R1, R2, R3, R4 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, yl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 1 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, yl, furanyl, ranyl, dinyl, imidazolyl, benzyl, isoPropyl, tertButyl, CH3; R8 and R9 are each independently selected from the group consisting in phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, or heteroaromatics selected from the group comprising pyrrolyl, l, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an r independently selected from 1 to 15; Z is H, N02, OH, NR10R11 Where R10 and R11 are each independently selected from the group consisting in H, (CH2)nCH3, NHCOR12 Where R12 is (CH2)nCH3, aromatic and heteroaromatics selected form the group comprising phenyl, naphthyl, pyrrolyl, fiaranyl, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 Where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, yl, fiaranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOzR14 with R14 is phenyl, 2- ,3- or 4, substituted phenyl, yl, heteroaromatics selected from the group comprising yl, l, thiofilranyl, dinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer independently selected from 1 to 15; W0 2013/093885 16 Formula VI wherein m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl—, Br- or 1-; R4, R15 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, CH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently selected from the group consisting in H, , 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, pyrrolyl, furanyl, thiofuranyl, dinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group consisting in phenyl, 2- ,3- or 4- tuted phenyl, 2- or 3-naphthyl, or heteroaromatics selected from the group comprising yl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the ipt n is an integer independently selected from 0 to 15; Z is H, N02, OH, NRlORll where R10 and R11 are each independently selected from the group consisting in H, (CH2)nCH3, NHCORl2 where R12 is (CH2)nCH3, aromatic and heteroaromatics ed form the group comprising phenyl, naphthyl, pyrrolyl, l, thiofilranyl, pyrimidinyl, imidazolyl, COORl3 where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, yl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOle4 with R14 is phenyl, 2- ,3- or 4, substituted , yl, heteroaromatics ed from the group comprising pyrrolyl, filranyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer independently ed from 0 to 15; Formula VII wherein m is an integer selected from 1 to 3; W is selected from H and halogens; the halogen is ed from F-, Cl—, Br- or 1-; R4 is H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, pyrrolyl, filranyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or 9; R5, R6 and R7 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, olyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group ting in phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, or aromatics selected from the group comprising pyrrolyl, furanyl, thiofuranyl, dinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer independently selected from 0 to 15; Z is H, N02, OH, NR10R11 where R10 and R11 are each independently selected from the group consisting in H, (CH2)nCH3, NHCOR12 where R12 is (CH2)nCH3, aromatic and heteroaromatics selected form the group comprising phenyl, naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising , naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOzR14 with R14 is phenyl, 2- ,3- or 4, substituted phenyl, yl, heteroaromatics selected from the group sing pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer independently selected from 0 to 15; R?1 X Formula VIII \Heteroaromatic—Z wherein the heteroaromatic is an aminopyrrole, aminofilrane, aminothiofurane, or a pyrimidine; R1, R2, R3, R4 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- tuted phenyl, 2- or 3-naphthyl, pyrrolyl, l, thiofilranyl, pyrimidinyl, olyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8, or NHSOZR9; where R5, R6, R7, R8, R9 are each ndently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, yl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, or (CH2)nCH3 the subscript n is an integer independently selected from 0 to 15; Z is H, N02, OH, NR10R11 where R10 and R11 are each ndently selected from the group consisting in H, (CH2)nCH3, NHCOR12 where R12 is (CH2)nCH3, aromatic and heteroaromatics selected form the group sing phenyl, naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 where R13 is H, CH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, NHSOzR14 with R14 is phenyl, 2- ,3- or 4, substituted , naphthyl, heteroaromatics selected from the group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer independently selected from 0 to 15; W0 2013/093885 18 lawX\Heteroaromatic—Z Formula IX wherein the subscript n is an integer independently selected from 1 to 15; the heteroaromatic is an aminopyrrole, urane, aminothiofurane, or a pyrimidine; R4, R15 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, pyrrolyl, furanyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, pyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, pyrrolyl, l, thiofuranyl, pyrimidinyl, olyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group consisting in phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, or heteroaromatics ed from the group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer independently selected from 0 to 15; Z is H, N02, OH, NR10R11 where R10 and R11 are each independently ed from the group consisting in H, (CH2)nCH3, 2 where R12 is (CH2)nCH3, aromatic and heteroaromatics ed form the group comprising phenyl, naphthyl, pyrrolyl, l, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 where R13 is H, (CH2)nCH3, ic and heteroaromatics selected from the group comprising phenyl, naphthyl, yl, filranyl, thiofilranyl, pyrimidinyl, olyl, NHSOzR14 with R14 is phenyl, 2- ,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from the group comprising pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer independently selected from 0 to 15; WX 11113 X \Heteroaromatic—Z wherein the heteroaromatic is an aminopyrrole, aminofilrane, aminothiofurane, or a pyrimidine; R4 is H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3-naphthyl, pyrrolyl, filranyl, ranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3 alkenyl, alkynyl; the subscript n is an integer independently selected from 0 to 15; X is O, S, CR5R6, NR7, NHCOR8 or NHSOzR9; R5, R6 and R7 are each independently W0 2013/093885 19 selected from the group consisting in H, phenyl, 2- ,3- or 4- substituted phenyl, 2- or 3- naphthyl, pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, isoPropyl, tertButyl, (CH2)nCH3; R8 and R9 are each independently selected from the group consisting in phenyl, 2- ,3- or 4- substituted , 2- or 3-naphthyl, or heteroaromatics selected from the group comprising pyrrolyl, furanyl, thiofuranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3; the subscript n is an integer ndently selected from 0 to 15; Z is H, N02, OH, NR10Rl l Where R10 and R11 are each independently selected from the group consisting in H, (CH2)nCH3, NHCORl2 Where R12 is CH3, aromatic and heteroaromatics selected form the group comprising phenyl, naphthyl, pyrrolyl, filranyl, thiofilranyl, pyrimidinyl, imidazolyl, COOR13 Where R13 is H, (CH2)nCH3, aromatic and heteroaromatics selected from the group comprising phenyl, naphthyl, yl, filranyl, thiofilranyl, pyrimidinyl, olyl, NHSOle4 with R14 is , 2- ,3- or 4, substituted phenyl, naphthyl, heteroaromatics selected from the group comprising yl, filranyl, ranyl, pyrimidinyl, imidazolyl, benzyl, (CH2)nCH3 ; the subscript n is an integer ndently selected from 0 to 15; Even more preferably, the tive having Notch signalling pathway inhibition properties is selected from the group consisting in Formula 11 d Non, 4-(4-(tert-pentyl)phenoxy)aniline, 00 Formula 111 a 4-(4-cyclohexylphenoxy)aniline, Formula IV a 6-(4-((3r,5r,7r)—adamantan— l -yl)phenoxy)pyridin-3 -amine, om Formula 11 e 6-(3-(tert-butyl)phenoxy)pyridin—3-amine, grow Formula 11 f 4-(4-(tert-butyl)phenoxy)-3 -fluoroaniline, 0*}N / Formula 11 g 6-(4-(tert-Pentyl)phenoxy)pyridin—3-amine, \AD/OfiN / Formula 11 h 6-(4-Butylphenoxy)pyridinamine, $0om Formula 111 b 4-(4-Cyclohexylphenoxy)fluoroaniline, 00 Formula 11 i 3-Fluoro(4-(tert-pentyl)phenoxy)aniline, omN / a 11 j 6-(4-(2—Methylpentan—2-yl)phenoxy)pyridin— 3 -amine, Formula IV b 4-(4-((3r,5r,7r)—Adamantan— l - noxy)aniline, 00 Formula IV c 4-(4-((3r,5r,7r)—Adamantan— l -yl)phenoxy)—3 - fluoroaniline, fixmy", \T/m“'\ “i3 N, Formula 111 c .\ : ‘ : §§ ‘ : : .J‘ x > \\‘ ,/ \\;{x \\\ r3” “’3“ ‘f“"\\ in I §] Formula 11 k \ ‘\. ,‘ 1"‘\ \ \ a" x. f“\ “a ‘ \ ¢ .‘g "x 9' NY! \NQ‘: \ ~ .
K Formula II 1 \ [§ l§I ,. : Mr“ R w \ .—\:k"'¢\x Fl? v V ‘5}5 Formula 11 m 6-(4-(2,4,4-trimethylpentan—2- yl)phenoxy)pyridin—3-amine.
W0 2013/093885 22 2012/057622 The invention also s to salts or solvates of the compound 13, chemical modified 13 compounds and derivatives of said 13 compounds of the invention. Preferably, these salts and/or solvates are pharmaceutically acceptable. According to the present invention, pharmaceutically acceptable salts are produced from acidic inorganic or organic nds, or alkaline inorganic or organic nds. As used herein, the phrase “pharmaceutically acceptable salt” refers to a salt that retains the biological iveness of the free acids and bases of a specified compound and that is not biologically or otherwise rable.
Unless specified otherwise, it is further understood that all isomers, including enantiomers, isomers, rotamers, tautomers and racemates of the compound 13, chemical modified 13 nds and derivatives of said 13 compounds of the invention are contemplated as being part of this invention. The invention includes stereoisomers in optically pure form and in admixture, including racemic mixtures. Isomers can be ed using conventional techniques, either by reacting optically pure or lly enriched starting materials or by separating isomers of compounds of the present invention.
“Racemates” refers to a mixture of enantiomers.
“Stereoisomer” or “stereoisomers” refer to nds that differ in the chirality of one or more stereocentres. Stereoisomers include enantiomers and diastereomers. The compound 13, chemical modified 13 compounds and derivatives of said 13 compounds of this invention may exist in stereoisomeric form if they s one or more asymmetric centres or a double bond with asymmetric substitution and, therefore, can be produced as dual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of Advanced Organic Chemistry, 4th ed., J. March, John Wiley and Sons, New York, 1992).
“Tautomer” refers to alternate forms of a compound that differ in the position of a proton, such as enol-keto and enamine ers, or the tautomeric forms of heteroaryl groups containing a ring atom attached to both a ring -NH- moiety and a ring =N-moiety such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.
W0 2013/093885 23 A skilled person will know that, if compound 13, al modified 13 compounds and derivatives of said 13 compounds of the ion contain charged group, a suitable counterion will be derived from an organic or inorganic acid. Such counterions include halide (such as chloride, bromide, fluoride, iodide), sulfate, phosphate, acetate, ate, citrate, lactate, maleate, te, palmitate, cholate, glutamate, glutarate, tartrate, stearate, salicylate, methanesulfonate, benzenesulfonate, sorbate, e, te, cinnamate, and the like. If the polar moiety is a negatively charged group, a suitable counterion will be ed from sodium, um, barium, calcium, copper, iron, lithium, potassium and zinc, and the like.
Surprisingly, the chemical compound ert-Butylphenoxy)Pyridin—3-Amine (13) was identified as a potential Notch inhibitor. Interestingly, 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) was found to block NICD mediated pathway activation (figure 1). Because of its ability to attenuate NICD mediated Notch activation, 6-(4-Tert-Butylphenoxy)Pyridin Amine (I3) is able to block proliferation ofNICD overexpressing leukemic cell lines which are resistant to DAPT (a-y—secretase inhibitor, N- [N-(3,5-difluorophenacetyl-Lalanyl)]-(S)- glycine t-butyl ester) (figure 5). The Notch inhibitory potential of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) was further confirmed by the downregulation ofNotch target genes in human T-ALL cell lines (figure 3) and Affymetrix geneChip array (data not shown). The fact that 6-(4-Tert-Butylphenoxy)PyridinAmine (13) can induce entiation of C2C12 cells into MHC expressing multinucleated myotubes further validated the anti-Notch role of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) (data not . The Notch pathway inhibition caused by 6-(4-Tert-Butylphenoxy)PyridinAmine ( 13) can be rescued by the overexpression ofMAMLl above certain levels. For example, 6-(4- Tert-Butylphenoxy)PyridinAmine (13) was able to block the signalling with 800 ng of NICD and 1 ug ofMAMLl was transiently introduced into the cells, however when the amount ofMAMLl was increased to 3 ug, 6-(4-Tert-Butylphenoxy)PyridinAmine ( 13) was no longer able to block the pathway activation (Figure 2). These data suggest that, 6-(4- utylphenoxy)PyridinAmine (13) may interfere with the Notch transcriptional tion complex thereby inhibiting the signalling activation. Microscopic studies by introduction of MAML at levels where 6-(4-Tert-Butylphenoxy)PyridinAmine (13) could still block the pathway activation showed that 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment does not impede co-localization ofNICD, MAMLl and CSL/RBP-jk in the sub- nuclear compartments (data not shown). Without being bound to theory, one of the possible isms of action of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) could be to disrupt W0 2013/093885 24 the recruitment of transcriptional coactivators to the core CSL/RBP-jk-NICD-MAMLI complex. Therefore, the status of onal coactivators involved in the formation of onal transcriptional activation complex still needs to be determined. Under logical conditions, following the formation of CSL/RBP-jk-NICD-MAMLI complex, CBP/p300 histone acetyltransferase (HAT) is recruited to the complex leading to its autoacetylation and acetylation of histone 3 and 4. 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13), as well as derivatives thereof, were fiarther igated in an in viva context to determine their Notch inhibitory as well as toxic side effects in the mice. Notch signalling is essential for the nance of normal homoeostasis in the intestine. Genetic ablation or pharmacological inhibition ofNotchl and Notch2 signalling in the intestine leads to goblet cell metaplasia in the intestine. Since, 6-(4- Tert-Butylphenoxy)PyridinAmine (13) has been observed to block Notchl and Notch2 ed signalling mice treated with 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) were expected to develop goblet cell metaplasia. Surprisingly, treatment of mice with 25 mg/kg of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) for 7 days (more than a month in case of xenotransplants) did not perturb inal homeostasis and without any indication of goblet cell accumulation (data not shown). This unexpected outcome could be due to two reasons.
One le explanation could be that the tration of 6-(4-Tert-Butylphenoxy)Pyridin- 3-Amine (I3) (25mg/kg) used is not sufficient to block Notch pathway activation in the intestine. However, a second more plausible explanation could be the differences in the composition of transcriptional activation complexes downstream ofNotchl and Notch2 signalling . Due to these possible differences, Notchl and Notch2 mediated signalling may have different sensitivities against 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment.
Notch signalling plays an important role in the regulation of hematopoietic system. For instance, tchl signalling is essential for T cell development in the thymus. Notch2 and MAMLl ed pathway activation is critical for Marginal Zone B (MZB ) cells development in the spleen. To address whether 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) can impair Notch dependent MZB cell development in the spleen, C57Bl6 mice were treated with 25 mg/kg of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) for 7 days and analyzed on day 8. Flow try analyses using antibodies against B220, CD21 and CD23 ed that 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment causes a reduction the percentage and absolute numbers ofMZB cells in the spleen (figure 7).
Anti-cancer activity of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) was investigated in transplant models for human diseases, namely T-cell leukemia and breast cancer. In these studies, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) has demonstrated a remarkable ability to slow down the progression and metastasis of very aggressive form of leukemic cell lines (figure 8). In on, in a inary study using breast cancer as a model of solid tumors, 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment has led to a block in tumor progression in the mice (figure 9).
The chemical compound 6-(4-Tert-Butylphenoxy)PyridinAmine (13) has shown ability to block NICD mediated signalling . ore this compound is useful in cancers where Notch driven tumors are resistant to y-secretase inhibitor treatment.
The present invention also provides a pharmaceutical composition sing 6-(4- Tert-Butylphenoxy)PyridinAmine (13) of formula I, or one of its tives having Notch signalling pathway inhibition properties as described herein, or pharmaceutically acceptable salts, solvates, tautomers, isomers thereof, and a pharmaceutically acceptable carrier. As to the appropriate carriers, reference may be made to the standard literature describing these, e.g. to chapter 25.2 of Vol. 5 of “Comprehensive Medicinal Chemistry”, Pergamon Press 1990, and to “Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete”, by HP.
Fiedler, Editio Cantor, 2002. The term “pharmaceutically acceptable carrier” means a r or excipient that is useful in preparing a pharmaceutical composition that is generally safe, and possesses acceptable toxicities. Acceptable carriers include those that are acceptable for nary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” as used in the specification and claims includes both one and more than one such carrier.
Optionally, the pharmaceutical composition of the present invention r ses one or more additional active agents selected among the non limiting group sing chemotherapeutic agents for treating . Such chemotherapeutic agents may be selected among the group comprising, for example, Altretamine, Bleomycin, Busulphan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, tin, Cladribine, Crisantaspase, Cyclophosphamid, Cytarabine, Dacarbazine, Daunorubicin , Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Idarubicin, Ifosfamide, ecan, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, latin, Pentostatin, Procarbazine, Streptozocin, Taco, Temozolomide, , W0 2013/093885 26 Tioguanine/Thioguanine, Thiotepa, Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine and Vinorelbine.
The compounds of the invention, namely the 6-(4-Tert-Butylphenoxy)Pyridin Amine (I3) and derivatives thereof, that are used in the ent and/or prevention of s can be incorporated into a variety of ations and medicaments for therapeutic administration. More particularly, one or more compound(s) as ed herein can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers, and can be formulated into preparations in solid, semi- solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, nts, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, ermal, intracranial and/or intratracheal administration. Moreover, the compound can be administered in a local rather than systemic manner, in a depot or sustained release formulation. The nds can be formulated with common excipients, ts or carriers, and ssed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered by the intramuscular or intravenous routes. The compounds can be administered transdermally, and can be formulated as sustained release dosage forms and the like. The compounds can be stered alone, in combination with each other, or they can be used in combination with other known compounds. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by nce. Moreover, for a brief review of methods for drug delivery, see, Langer, Science (1990) 249: 1527-1533, which is orated herein by reference.
The amount of a compound as provided herein that can be combined with a carrier material to produce a single dosage form will vary depending upon the e treated, the subject in need thereof, and the particular mode of administration. However, as a general guide, le unit doses for the compounds of the present invention can, for example, preferably contain between 0.1 mg to about 1000 mg, between 1 mg to about 500 mg, and between 1 mg to about 300 mg of the active nd. In another example, the unit dose is between 1 mg to about 100 mg. Such unit doses can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total dosage for a 70 kg human adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration. A preferred dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of s including the activity of the specific compound ed; the age, body , general health, sex and diet of the individual being treated; the time and route of stration; the rate of excretion; other drugs that have usly been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area. A typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time- release capsule or tablet taken once a day and containing a proportionally higher t of active ingredient. The time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release. It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art.
The present invention fiarther provides a compound of the invention for use in treating and/or preventing cancers.
As used herein, cancers are preferably Notch dependent cancers and are selected from the non limiting group comprising T cell-Acute lymphoblastic ia (T-ALL), c myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), Mantle cell lymphoma, breast cancer, pancreatic cancer, prostate cancer, ma, brain tumors, tumor enesis, and colorectal cancer.
Preferably, the compounds of the present invention Tert-Butylphenoxy)Pyridin Amine (13), its derivatives) can be also used in the treatment of cancers where Notch dependent cancers are resistant to y—secretase inhibitor treatment. Notch signalling dependent human tumors resistant to y-secretase inhibitor treatment can be determined by the levels of NICD, Notch target genes as well as by mutation status ofNotch or and other components of the Notch pathway.
W0 2013/093885 28 The present invention also provides a method for treating and/or preventing cancers, said method comprising administering the 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13), its derivatives, or the pharmaceutical composition of the invention to a subject in need thereof.
In another ment, the present invention provides a method of treatment of a disease associated with an up-regulated Notch signalling pathway ty, said method comprising administrating the 6-(4-Tert—Butylphenoxy)PyridinAmine (13), a derivative thereof, or the pharmaceutical composition of the invention to a subject in need thereof.
The daily dose of compounds of the present invention will necessarily be varied depending upon the host treated, the particular route of administration, and the ty and kind of the illness being treated. Accordingly the optimum dosage may be determined by the tioner who is treating any particular patient. Further, it is noted that the clinician or treating physician will know how and when to start, interrupt, adjust, or terminate therapy in conjunction with individual patient response.
For any compound used in the method of the present invention, a therapeutically effective dose can be estimated initially from cell culture assays, animal models, or osing of human subjects.
“Treatment” as used herein, refers to both therapeutic ent and prophylactic or tative es. Subjects in need of treatment include those already with the disorder, such as , as well as those in which the disorder, such as cancer, is to be prevented.
Hence, the mammal, preferably human, to be treated herein may have been diagnosed as having the er, such as cancer, or may be posed or susceptible to the disorder, such as cancer.
The term “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the eutically effective amount of the drug may reduce the number of tumor or cancer cells, reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cells infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the compounds of the present invention may t growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The phrase W0 2013/093885 29 “therapeutically effective amount” is used herein to mean an amount ient to prevent, or preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells, or other e of pathology.
Optionally the compounds of the present ion may be used against cell proliferate diseases in combination (for example either at the same time, or almost at the same time, or one after the other) with conventional treatments such as standard radiotherapy and/or standard chemotherapy. The standard herapy and chemotherapy can be also the concomitant chemo-radiotherapy.
Therefore, optionally, the standard radiotherapy and/or chemotherapy can be performed , simultaneously or after the administration of a therapeutically effective amount of the compound of the present invention, or ceutical compositions ning thereof.
The term “concomitant chemo-radiotherapy” is used when these two treatments (chemotherapy and radiotherapy) are given either at the same time, or almost at the same time, for instance one after the other, or on the same day, etc.
The term “standard radiotherapy” refers to the use of ionizing ion as part of cancer treatment to control malignant cells. Preferably the ionizing radiation is diation. It is also common to combine radiotherapy with surgery, chemotherapy, hormone therapy, or combinations thereof. Most common cancer types can be usually treated with radiotherapy.
The e treatment intent (curative, adjuvant, neoadjuvant or palliative) will depend on the tumor type, location, and stage, as well as the general health of the subject in need thereof.
The term “standard chemotherapy”generally refers to a ent of a cancer using specific chemotherapeutic/chemical agents. A chemotherapeutic agent refers to a pharmaceutical agent generally used for treating cancer. The chemotherapeutic agents for treating cancer include, for example, Altretamine, Bleomycin, han, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Crisantaspase, Cyclophosphamid, Cytarabine, azine, Daunorubicin , Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, abine, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan, W0 93885 30 Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Pentostatin, Procarbazine, Streptozocin, Taco, lomide, , Tioguanine/Thioguanine, Thiotepa, Topotecan, Treosulfan, stine, Vincristine, Vindesine or Vinorelbine.
When a chemotherapeutic agent is used in combination with a nd according to the present invention, then this may be used in the form of a medicament containing a ation of these two agents, for simultaneous administration, or they may be used in the form of separate dosage forms, each containing one of the agents, and in the latter case the individual dosage forms may be used e. g. sequentially, i.e. one dosage form with the compound of the invention followed by a dosage form containing the chemotherapeutic agent (or vice versa). This embodiment of two te dosage forms may be conceived and provided in the form of a kit.
Also optionally the compounds of the present invention may be used against cell proliferate es, such as cancers, in combination with conventional removal of a tumor bulk, by for e segmental resection (biopsy or gross resection).
The term “removal of a tumor bulk” refers to any removal, ablation or resection of a tumor bulk from a subject. The removal can be chemical, radiation or surgical. Preferably said removal is surgical, such as ablation or ion. Resection can be “segmental resection” (or segmentectomy), a surgical procedure to remove part of an organ or gland from a subject. It may also be used to remove a tumor and normal tissue around it. Debulking agent may be also used to remove tumor bulk. The term “debulking agent” includes any molecule (e.g. chemical, biological) or any extemal/environmental agent (e.g. y—irradiation) or ional surgery that would allow killing cancer cells from the tumor bulk (e.g. FLl0 and FLl' cells as mentioned above).
Another object of the present invention is a kit sing one or more doses of 6-(4- Tert-Butylphenoxy)PyridinAmine (13), or of one of its derivatives having Notch signalling pathway inhibition properties, or the pharmaceutical composition of the present ion for use in a method for treatment and/or prevention of cancers. The kit can further comprise one or more doses of a chemotherapeutic agent. Optionally, the kit may also comprise reagents and/or instructions for use.
W0 2013/093885 31 Generally, the kit comprises a container and a label or e insert on or associated with the container. Suitable containers include, for e, bottles, vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds the pharmaceutical composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic ion needle). The label or package insert tes that the ition is used for treating the condition of choice, such as cancer.
The present invention also relates to the use of the compounds of the invention for inhibiting in vitro or in vivo the Notch signalling y in cells. Usually, said cells are cancer cells.
Also envisioned is a method of treating a subject for Notch ent cancer, comprising i) determining in cancer cells obtained from a biological sample of said subject whether the cancer is Notch signalling pathway dependent, ii) and treating said subject based upon whether the cancer is Notch dependent cancer by administering a therapeutically effective amount of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) of Formula I or one of its tives having Notch signalling pathway tion properties, or a pharmaceutical composition of the invention.
Usually, the Notch signalling pathway dependency in cancer cells is determined by any method known in the art. As an example, this method can consist in an in vitro y-secretase complex activity assays as described herein.
This method of treating may further comprise administering at least one conventional cancer treatment. The conventional cancer treatment is administered before, simultaneously or after the administration of the therapeutically effective amount of ert-Butylphenoxy)Pyridin— 3-Amine (13) of Formula I or one of its derivatives having Notch signalling pathway tion properties, or the pharmaceutical composition of the invention.
Usually, the conventional cancer treatment consists in radiotherapy and/or chemotherapy.
W0 2013/093885 32 The present invention also relates to the use of the compounds of the invention in a method for provoking apoptosis in a cell, either in vitro or in vivo, by inducing GO/Gl cell cycle Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention es all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or tively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not ctive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
Various references are cited throughout this Specification, each of which is orated herein by reference in its ty.
The foregoing ption will be more fully understood with reference to the following Examples. Such Examples, are, r, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.
W0 2013/093885 33 EXAMPLES Constructs and gene reporter : Mouse DL4-IRES dsRED cDNA was cloned into a pENTRl vector (Invitrogen®) and finally shuttled into a destination lentivirus vector using Gateway cloning gy (Invitrogen). A Phosphoglycerate kinase (PGK) promoter drove the expression of DL4 protein. DL4-lentiviral particles were produced in 293T cells by cotransfection of DL4- lentivirus , Gag/pol expression plasmid and plasmid ng for viral envelope proteins. To overexpress Notchl protein, mouse fiJll length Notchl cDNA was a cloned in a pCDNA3. -puromycin vector. Notchl cDNA was cloned upstream of IRES- puromycin between HindIII and XbaI restriction sites. A CMV promoter controlled the expression ofNotchl protein.
To measure the Notch signalling activation, CSL/RBP-jk consensus DNA binding sequences were cloned in a head to tail mation in the pGL4 luciferase vector (Promega), thus named leCSL/RBP-jjk luciferase vector. In order to determine the Notch y activation in al nd screening assay, 12x CSL/RBP-jk consensus DNA binding sequences were cloned into pGL4.26.luciferase vector (Promega). As an internal control for transfection efficiency, SV40 Renilla vector was used (Promega).
NICD-GFP overexpression studies were performed using pEGFP-Cl-NICD expression d. The FLAG-CMV2 plasmid expressing MAMLl-FLAG was a kind gift from Dr. Lizi Wu, Harvard Medical School, Boston.
Generation of DL4 and N1 stable HeLa cells: In order to generate DL4 and N1 stable cell lines, HeLa cell were bought from ATCC (catalog # CCL-2). To generate DL4 stable lines, cells were transduced with DL4-lentiviral particles. DL4 stable clones were selected using puromycin. High DL4 expressing clones were sorted using antibodies against DL4 with scence activated cell sorting (FACS).
To generate N1 stable line, cell were transfected with a pCDNA3. 1+ (Invitrogen) plasmid containing mouse full length Notchl under the control of CMV promoter. To select for Notchl expressing clones, an IRES puromycin cassette was cloned downstream ofNotchl cDNA. HeLa cells expressing high levels ofNotchl were enriched by FACS using anti- W0 2013/093885 34 2012/057622 Notchl antibodies. DL4- and Nl-HeLa cells were cultured in DMEM (GIBCO, ogen), % FCS and lOug/ml ofpuromycin (Sigma).
High-Throughput Screening and data es: For chemical library screen, the coculture assay was performed as s. Nl-HeLa cells were sfected in 10 cm tissue e dishes with 16 ug of pGL4.26. leCSL.luciferase vector/plate, 4 ug hl expression plasmid/plate, and 200 ng of SV40 Renilla vector/plate. DL4- and Nl-HeLa cells were detached from the plate by using 0.5mM EDTA (lXPBS). The both cell populations were counted and mixed in 1:1 ratio (5,000:5,000 well in a 384 well plate) and dispensed into 384 well plates (white, clear bottom, Corning) using multidrop Combi plate dispenser. The assay plates were pre-dispensed (using automated Biomek 3000 liquid handler) with chemical compound libraries (Microsource NIMDS, Maybridge Hitfinder and Prestwick) to give a final concentration of 10 uM. The final assay volume was 22 uls. Twenty-four hours later, growth media was aspirated and cells were lysed with lx Passive lysis buffer for 10 minutes at room temperature.
Luciferase activity was measured using Luciferase Assay Reagent II and Renilla values were determined by using Stop and Glow reagent (Dual luciferase assay , Cat # E1980, Promega). Luciferase and renilla readouts were taken using Tecan® F500 (Tecan) multiplate reader. All the liquid handling steps (aspiration of the medium, dispensing of Passive lysis , Luciferase Assay Reagent II and Stop and Glow reagents) were performed using ELF406 liquid handler.
Data analyses were med using in-house built analyses software at Biomolecular Screening Facility (BSF) at Ecole Polytechnique Federale de Lausanne (EPFL).
RNA extraction Total RNA was extracted from cells using TRIzol® extraction kit (Invitrogen).
Briefly, 1x106 cells were washed with ice-cold leBS and lysed in 1 ml of TRIzol® solution for 5 minutes at room temperature to dissociate nucleoprotein complexes. Lysed cells were then treated with 200 ul of chloroform and shaked vigorously for 15-30 s and incubated at room temperature for 2-3 minutes. The samples were centrifuged at 14000 rpm using Eppendorf table top centrifuge for 10 minutes at 4°C. Following centrifilgation, upper aqueous phase was transferred to new eppendorf tubes. To precipitate total RNA 500 ul of isomyl alcohol was added to the separated aqueous phase and incubated at room temperature for 10 minutes. A RNA pellet was obtained by centrifilging the samples at 4°C for 10 minutes. RNA pellet obtained was washed with 1 ml ice cold 75% ethanol and spun down at 14000 rpm at 4°C. RNA pellet was dried off of excess of ethanol and resuspended in 40 ul DPEC water. cDNA synthesis: Total RNA ted from the cell was used to size cDNA by e transcription reaction. Reverse transcription was performed using SuperScriptTM RT (Invitrogen). RNA concentration was measured using NanoDrop®ND-1000 spectrophotometer (Witec AG) and 500 ng of total RNA was mixed with a 10mM mix of dNTPs and 100 ng ofrandom primers. The reaction mix was incubated at 65°C for 5 minutes and quickly ted on ice for 1 minute. Following incubation on ice, 5x first strand buffer and 0.1M DTT were added and mix was incubated for 2 minutes at 25°C. To start the reverse transcription reaction, 200U of SuperScripTM 11 RT was added to the reaction mix and incubated at 42°C for 50 minutes. The reaction was stopped by incubating the reaction mix at 75°C for 15 s.
Western blot analyses: Cells were lysed in RIPA buffer (50mM Tris.Cl, pH 7.5, 150 mM NaCl, 1% t P-40, 0.5% sodium deoxycholate and 0.1% SDS) for 30 minutes at 4°C. Lysed cells were fuged to remove the debris at 14000 rpm at 4°C. Supernatant was transferred to a new eppendorf tube. The protein concentration was determined by Bradford assay using spectrophotometer (Ultrospec 3000 pro). 40 ug of protein were denatured in 1x SDS gel loading buffer (100mM Tris.Cl, pH 6.8, 200 mM DTT, 4 % SDS, 0.2 % bromophenol, 20% glycerol) by heating at 99°C for 5 minutes. Denatured protein samples were stored on ice until loading on to the acrylamide gel. The samples were run on 8% or 10% acrylamide gel in Trisglycine ophoresis buffer (25mM Tris, 250 mM glycine, 0.1% SDS). Following separation on the acrylamide gel, protein samples were transferred on to PVDF membrane (PEQ lab, catalog number 39-3010) using transfer buffer (39mM glycine, 48 mM Tris base, 0.037 % SDS and 20 % methanol).
For immunoblotting, membranes were blocked with 5 % milk and incubated overnight with primary dies at 4°C. Membrane were washed with 1x TBST (1x TBS + 0.5 % tween 20) for 5 s (3 times) and incubated with HRP-conjugated secondary antibodies for one hour at room temperature. Signal was detected with Super Signal West chemiluminescent substrate o Scientific, catalog number 34077).
W0 2013/093885 36 Immunofluorescence staining: To perform immunofluorescence staining, HeLa cells or C2C12 cells were grown on cover slips. Cells were washed with 1X ice-cold PBS, fixed with 4% PFA for 5 minutes at room temperature and permeabilized using 0.3% Triton X-100. Subsequently permeabilized cells were blocked for 20 minutes with 1 % BSA for 20 s at room temperature. Cells were incubated with appropriate primary antibodies for one at room temperature. Alexa Fluor- 488 conjugated secondary antibodies were used to detect primary antibodies. Cells were counterstained with DAPI and mounted in fluorescent mounting media. scent images were Viewed and captured using Zeiss an microscope at Bioimaging and optics core facility at EPFL.
Table 1: List of the antibodies and g dilutions.
Anti-Val 1744 NICD WB and ChIP 1:1000 Cell Signal, 2421S Anti-Notchl (C-20) 1:1000 Santa Cruz, sc-6014 Anti-RBP-jk 1:500 Santa Cruz, sc-28713 Anti-FLAG-M2 1:500 Sigma, F1804 Anti-Hes1(H-140) 1:500 Santa Cruz, sc-25392 Anti-cMyc (9E10) 1:500 Abcam, ab11917 elta like 4 Flow try 1:100 Produced se Anti-Notchl Flow cytometry Produced in—house Anti-Myosin heavy chain 1:200 Sigma, MY-32 HRP-conjugated anti-goat IgG 1:3000 Invitrogen, 611620 HRP-conjugated anti-mouse WB 1:3000 GEhealthcare,NA931V HRP-conjugated anti-rabbit WB 1:3000 GEhealthcare,NA934V Alexa Fluor-488 secondary Ab 1:1000 Anti-B220 -Pacific blue Flow cytometry 1:400 Produced in—house Anti-CD21-FITC Flow cytometry 1:200 Anti-CD23-PE Flow cytometry 1:400 BD Pharmingen CR B-APC eF780 Flow cytometry 1:400 Anti-CD4-FITC Flow cytometry 1:800 Produced in—house Anti-CD8-Alexa 648 Flow cytometry 1:600 Produced in—house Anti-CD71-PE Flow cytometry 1:800 Anti-Ter119-APC eF780 Flow cytometry 1:200 Anti-AnnexinV-CyS Flow cytometry BD Pharmingen C2C12 Myoblast differentiation assay: C2C12 cells were grown on collagen-coated cover slip in the presence of growth media (10% serum). To induce myoblast differentiation, cells were grown to 100 % confluency for 3 days in the presence of differentiation media (2% horse serum) or in the presence of growth media + Notch inhibitors. After 3 days, cells were washed with 1x ice- cold PBS and fixed with 4 % PFA. fluorescence staining was performed using anti- MHC antibody as explained in the n 2.2.6 (Immunofluorescence staining).
Flow cytometry analyses: scence activated cell sorting (FACS) analyses were performed on CyAnTM ADP instrument platform for flow try at Flow cytometry core facility, EPFL. DL4 and Notchl expression in DL4- and Nl-HeLa cells was determined using anti-DL4 and anti-N1 antibodies respectively. T cell development in the thymus was investigated using antibodies against CD4, CD8 and TCRB. MZB cell development was monitored using antibodies against B220, CD21 and CD23. In brief, a single cell suspension was prepared from thymus and spleen. 1x106 cells suspended in 50 ul of staining media (HBSS supplemented with 2% NCS and 25mM HEPES) and stained with appropriate dy combinations by incubating on ice for 30 s.
To quantitate the percentage of apoptotic cells, AnnexinV and 7AAD staining was performed. Thymic cells were suspended in 300 ul of 1x nV binding buffer (BD Biosciences, San Diego, USA) and incubated with 10 ul of AnnexinV-CyS antibody and 10 ul of 7AAD (BD Biosciences, San Diego, USA). Samples were ted for 15 minutes at room temperature. FACS was med with in one hour of antibody staining.
Flow cytometry analyses were done on live cells by gating on d scatter (FSC) and side scatter (SSC). Data were analyzed by FlowJo software (Tree Star, Ashland, OR).
W0 2013/093885 38 Alamar blue proliferation assay: Alamarblue® proliferation assays were med to determine the growth kinetics of Notch inhibitor treated cells. Alamar blue® consists of a cell ble substrate resazurin. In metabolically active and proliferating cells, resazurin is converted to resorufin due to an intrinsic reducing power of live cells and produces a red cence. Therefore production of resorufin serves as an indicator of the viability of the cell population.
Proliferation assays were performed by seeding 5000 cells/well in a 96 well plate.
Cells were treated with DMSO or Notch inhibitors for different time intervals. Each treatment for every time interval was d out in 8 replicates. To determine the growth cs, 10 ul ofAlamar blue® (lnvitrogen) was added to each well and incubated for 4 hours. Alamarblue t was taken using Tecan F500 (Tecan) multiplate reader.
Haematoxylin & Eosin staining: Organs were harvested, fixed in 4% paraformaldehyde (PFA) overnight at 4°C and embedded in paraffin. Tissue sections were dewaxed and hydrated using decreasing tration of ethanol (100%-70%) and finally in led water. ns were stained with Hemotoxylin for 5 minutes, rinsed in acid alcohol for about 20 seconds and then rinsed in running water for 10 minutes. ns were then stained with Eosin for 5 minutes, washed in water and dehydrated using increasing concentration of l (70%-100%) and cleared in xylene solution. Sections were the mounted using mounting solution. Haematoxylin and Eosin stained sections were viewed and images were captured using Leica DMI4000 microscope.
Alcian blue staining: Intestinal tissue was flushed with ice-cold leBS, fixed in 4% PFA. Tissues were embedded in paraffin and sectioned to a thickness of 4 microns. Intestinal sections were deparaffinized at 60 OC and hydrated with decreasing concentration of alcohol (100% -70%) and finally washed in distilled water. Alcian blue staining was performed for 30 minutes at room temperature washed in running water and finally counterstained in nuclear fast red solution for 5 minutes. Tissue sections were then washed in running water ated in 100% alcohol and cleared in xylene solution. Mounted sections were then viewed and images were captured using Leica DMI4000 microscope.
W0 2013/093885 39 Experimental mice: Mice were kept and bred at Animal ty, EPFL, Lausanne. C57B16 mice were used to assess the intestinal toxicity of the chemical compounds. MMTV-ErbB2/Neu-IRES Cre (FVB background) mice were ed from Dr. William J Muller, McGill University, Montreal and genotyped using MMTV-ErbB2/Neu specific primers i-Siegel et al., 2008). NOD/SCIDyc '/' mice were bought from The Jackson Laboratory (USA) and were kept and bred at Animal facility, EPFL, Lausanne.
Intestinal toxicity and effect on Marginal Zone B cell development: C57Bl6 mice were intra peritoneal (i.p) injected with oil or 25 mg/kg of 13 or 10 mg/kg of CPA, once a day for 5- 7 days. Mice were weighed using a weighing scale on day 0, day 3 and day 5. On day 8, intestinal tissue, spleen and thymus were harvested for es.
Tumor transplantation assay: The human leukemic cell lines RPMI 8402 and HPB ALL were transduced with a lentivirus containing luciferase gene constitutively expressed downstream of a CMV promoter. The human leukemic cell lines RPMI 8204 (0.5-1 x106 cells) and HPB ALL (1 X106) were suspended in 100 ul of ice-cold leBS and kept on ice until the transplant.
NOD/SCIDyc '/' mice were lanted with the human leukemic cell lines by intravenous (iv) injection. Mice were monitored for tumor development using Caliper IVIS (Xenogen) live imaging system. Briefly, the luciferase substrate luciferin (Biosynth, L-8820) was ved in leBS and was injected (intra peritoneal) into the mice at a tration of 150mg/kg ofbody weight. Mice were imaged 5 minutes after the luciferin injection using Caliper IVIS live imaging system.
At day 13-15, mice were treated with oil or 25 mg/kg of 13 on a daily basis. Images were captured at the end of the ments.
Primary MMTV-ErbB2/Neu y tumors were harvested from the mice and a single cell suspension was ed. 1 xlO6 primary tumor cells were suspended in 50 ul of 1x PBS and kept on ice. Three weeks old recipient FVB mice were cleared of their endogenous epithelium and tumor cells were injected into the empty fat pad. Tumor development in the recipient mice was monitored and tumor volumes were measured using digital caliper. Tumor volumes were calculated using following formula: 2 x length x (width)2. Once the tumor reached a volume of about 100 mm3 , recipient mice were treated with oil or 25 mg/kg of 13 on alternate days.
W0 2013/093885 40 Assay development In order to identify novel modulators of the Notch pathway, Applicants have established a coculture assay in which DL4 ligand expressing HeLa cells were cultured with N1 HeLa cells, thereby activating the Notch pathway. The use of a DL4 and N1 HeLa cell coculture system mimics physiological conditions of ell communication between ligand and receptor sing cells. The in vitro generation of a controlled receptor-ligand assay system allowed Applicants to modify and monitor the Notch signal intensity by y-secretase tors .
DL4: N1 HeLa cell coculture activates Notch signalling To set up a coculture assay, Applicants have established DL4 and N1 expressing stable HeLa cell lines. In brief, HeLa cells were transduced with a lentivirus containing DL4 cDNA downstream of the PGK promoter. The D4 expressing cell population was enriched by fluorescence activated cell sorting. Similarly, the N1 stable HeLa cell line was established using a plasmid containing the mouse Nl cDNA followed by an IRES Puromycin selection cassette. This system allowed Applicants to select for only Notchl expressing clones when selected using puromycin. The expression levels of DL4 and N1 proteins in the tive cell lines were detected using anti-DL4 and anti-Nl antibodies. fication of the n levels by flow cytometry showed high level expression of DL4 and N1 compared to parental HeLa cells (data not shown).
To assess the Notch pathway activation ial of the stable cell lines, DL4 and N1 stable HeLa cells (DL4-HeLa and Nl-HeLa, tively) were cocultured in 1:1 ratio in a 6-well plate and grown to confluency. The cocultured cells were treated with DMSO or DAPT (lO uM) for 24 hours. For comparison, parental HeLa cells were also cocultured with DL4- HeLa cells and were grown in the presence or e of DAPT for 24 hours. Western blot es for the active form ofNotchl (NICD) using VALl744 antibodies was performed and ed only modest levels ofNICD when parental HeLa cells were cocultured with DL4 HeLa cells (data not shown), accounting for a low level of endogenous Notchl in HeLa cells.
On the other hand, in the absence of ligand (DL4-HeLa cells) or in the presence of GSI (DAPT) NICD levels were not detected, indicating a loss ofNotch signalling (data not shown). However, cocultures of DL4- and a cells revealed significantly higher levels ofNICD, which can be blocked by DAPT treatment (data not shown). Transient introduction W0 93885 41 of full length Notchl cDNA into Nl -HeLa cells further enhanced the robustness of the coculture assay as indicated by the increased levels ofNICD protein (data not shown). ting the N1 cleavage with DAPT can te the increase in Notch signalling activity (data not shown). These results confirmed that high levels of the Notch pathway activation could be achieved in the DL4:Nl coculture assay that responds to GSI inhibition.
The establishment of DL4:Nl coculture assay in a 6 well plate format allowed Applicants to assess receptor-ligand interaction mediated Notch signalling activation. GSI (DAPT) treatment of the coculture system can block receptor-ligand interaction driven Notch signalling .
Establishment of High-through put screening (HTS) ible assay lly, DL4:Nl coculture assay was established in a 6-well plate. In order to set up a high-through put screen (HTS), the assay system was fiarther optimized to robustly work in a 384 well plate format..
The assay was scaled-down to a 384 well plate format for screening of chemical compound libraries . In order to accomplish this, Nl-HeLa cells were transfected with a reporter plasmids and N1 expression vector. Twelve hours later, chemical compounds were sed into a 384 well plate along with DMSO and DAPT as negative and positive controls. DL4- and a cells were mixed in a 1:1 ratio (5000: 5000 cells/well) and added to 384 well plates using multidropCombi plate dispenser. Luciferase readout was measured using dual luciferase assay system. To optimize and determine the reproducibility of the assay, half of the plate was d with DMSO (192 wells) and the second half was treated with 10 uM DAPT (l92 wells). DAPT treatment led to a 10-fold downregulation ofNotch signalling activation.. The Z’ value for this assay was higher than 0.5. A Z’ value of >0.5 confirms the reliability and reproducibility of the assays for a HTS campaign.
Example 2 6-(4-Tert-Batylphen0xy)PyridinAmine (13) as a novel Notch signalling inhibitor 13 inhibits NICD mediated tion of Notch signalling : To validate the Notch inhibitory activity and ine the IC50 value of the I3 compound, the DL4:Nl coculture assay system was used. The cells in the coculture assay were treated for 24 hours with an sing concentration of I3 (2 -10uM). The activation of the Notch pathway was measured using a Notch driven luciferase reporter assay. As shown in W0 2013/093885 42 figure 1A, 13 blocks Notch signalling in a concentration dependent manner with an ICSO value in the lower uM range.
To determine whether over expression ofNICD can rescue I3 mediated inhibition of Notch signalling HeLa cells were co-transfected with a NICD expression plasmid and leCSL rase construct. The transfected cells were treated with an sing concentration of 13 and DAPT. Surprisingly, treatment ofNICD expressing cells with 13 could block y activation in a dose-dependent manner, while DAPT had no effect on the signalling activation (Figure 1B). This data suggests that 13 mediated inhibition of the Notch pathway is due to its activity downstream of the S3 cleavage event.
Next Applicants investigated r 13 could block pathway activation via other Notch receptors or is it c to Notchl signalling . To address this, a coculture assay was used where Notch signalling was ted via DL4:Nl or DL4:N2 ligand receptor pairs. The treatment of cells in these two coculture assays with 13 caused an inhibition ofNotch signalling via both DL4:Nl and DL4:N2 ligand-receptor pairs (Figure 1C). Similarly, 13 could also block pathway tion by Notchl-intracellular domain (NICD) and Notch2- intracellular domain (N2-ICD), suggesting that 13 is not specific for NICD mediated activation (Figure 1D). 6-(4-Tert-Butylphenoxy)PyridinAmine does not block nuclear zation of NICD: In vitro data from NICD transfected HeLa cells suggested that 6-(4-Tert- Butylphenoxy)PyridinAmine (13) blocks Notch signalling by acting downstream of S3 cleavage event. This raises several possibilities about the mechanism of action of 6-(4-Tert- Butylphenoxy)PyridinAmine (13). For example, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment could impair nuclear localization of the NICD. A second possible mechanism of inhibition could be targeting of one or more individual components of the transcriptional activation complex in the nucleus. To test whether 6-(4-Tert-Butylphenoxy)PyridinAmine (13) has an impact on nuclear transport ofNICD, HeLa cell were transfected with a NICD- GFP fusion construct and treated with DMSO and 6-(4-Tert-Butylphenoxy)PyridinAmine (13). This allowed Applicants to follow ort of fusion protein within the cell. In parallel, the 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated pathway inhibition was determined by Notch driven luciferase measurement (data not . Microscopic studies showed that in DMSO treated cells NICD-GFP fusion protein ocate to the nucleus, W0 2013/093885 43 which was not perturbed upon 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment (data not shown). This data rule out nuclear exclusion ofNICD as a mechanism of action of 16-(4- Tert-Butylphenoxy)PyridinAmine (13).
Over expression of MAMLl above a certain threshold can rescue 6-(4-Tert- Butylphen0xy)PyridinAmine (I3) induced Notch signalling inhibition: As 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) mediated blockage ofNotch signalling does not involve nuclear exclusion ofNICD, Applicants addressed whether 6-(4- Tert-Butylphenoxy)PyridinAmine (13) blocks interaction and thereby sub-nuclear localization ofNICD, MAMLl and P-jk (all parts of the core transcriptional activation complex). To resolve this, HeLa cells were co-transfected with 800 ng ofNICD- GFP plasmid, 1 ug of MAMLl-FLAG expression vector and grown on cover slips. The transfected HeLa cells were treated with DMSO or ert-Butylphenoxy)Pyridin—3-Amine (I3) (lOuM) for 24 hours. The ability of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) to block Notch activation at this concentration ofNICD and MAMLl was verified by Notch driven luciferase measurement (data not . Following treatment, the cells were fixed with 4% PFA, blocked with 1% BSA and d with antibodies against FLAG tagged MAMLl and CSL-RBP-jk. NICD-GFP filSlOI‘l n was Visualized by tracing the GFP protein. When expressed alone, NICD-GFP protein was localized in the nucleus in a diffilsed manner and ert-Butylphenoxy)Pyridin—3-Amine (13) treatment did not alter its nuclear localization. However overexpression ofNICD-GFP and MAMLl led to co-localization of both proteins into sub-nuclear tments (possibly nuclear bodies). The 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treatment of the cells did not perturb the translocation and co-localization ofNICD-GFP and MAMLl into sub-nuclear compartment (data not shown).
Similarly, the location of CSL/RBP-jk was igated using antibodies specific t this protein. As shown in figure 22C, MAMLl-FLAG and CSL/RBP-jk co-localized in sub- nuclear compartments and 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment did not perturb their distribution in the nucleus (data not shown). This data suggest that 6-(4-Tert- henoxy)PyridinAmine (13) treatment does not perturb co-localization of components of the Notch transcriptional activation x in the nucleus. However, whether it blocks interaction between various components of the x still need to be investigated.
W0 2013/093885 44 To fiarther investigate the mechanism of action of 6-(4-Tert-Butylphenoxy)PyridinAmine (13), Applicants speculated that 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) might be targeting one of the components of the transcriptional activation complex. The over expression of this target protein may titrate out 6-(4-Tert-Butylphenoxy)PyridinAmine (13) compound and thus rescue 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) induced pathway inhibition. To address this question, HeLa cells were co-transfected with 800 ng ofNICD- GFP plasmid and with an increasing amount (0, l and 3 ug) of MAMLl-FLAG expression vector. The Notch pathway activation was measured by introducing l2xCSL luciferase plasmid. As shown in figure 2, in HeLa cells transfected with NICD alone or NICD + 1 ug of MAMLl treatment can block Notch ling , 6-(4-Tert-Butylphenoxy)PyridinAmine (13) in a concentration dependent manner. However, when the amount of MAMLl plasmid was increased to 3ug, 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment was no longer able to t the activation of Notch signalling (Figure 2). Therefore, over expression of MAMLl above a certain threshold could rescue 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated inhibition of the Notch y. This data suggest that MAMLl itself might be the target of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) compound. An increase in the concentration ofMAMLl may be able to titrate out the inhibitor and thus ing it incapable of blocking the signalling cascade. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment decreases Notch signalling in human cancer cell lines: Aberrant tion ofNotch ling plays an ant role in tumor initiation and/or nance of human cancers. To determine whether 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treatment can block Notch signalling in human cancer cells, various cancer cell lines (T-ALL cell lines RPMI 8402, HPBALL, KOPTKl and pancreatic cancer cell line PANC l) were treated with 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) for 24 hours. The effect on Notch signalling was determined by measuring the expression levels ofNotch target genes. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment of human cancer cell lines (RPMI 8402, HPBALL, KOPTKl and PANCl) for 24 hours and uent analyses h target genes by qRT-PCR or Western blot analyses showed that 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) induced a statistically significant downregulation ofNotch target genes such as Hesl, cMyc and Dtxl at the mRNA as well as at the protein levels (Figure 3). The downregulation of Notch target genes correlates with reduced levels ofNICD (Figure 3B, C and D).
As treatment of the human T-ALL cell lines and PANCl pancreatic cancer cell line with 6-(4- Tert-Butylphenoxy)PyridinAmine (I3) induced a downregulation ofNotch signalling , ants questioned whether this inhibition of the pathway translates into growth arrest in cancer cells. To this end, RPMI 8402, KOPTKl, PANCl and nRas driven melanoma cell lines were grown in the presence or absence of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) for several days and their proliferative index was ed using the Alamar blue assay. In addition, B-lymphocyte RAJI cell lines with no known Notch mutations were used as a control (data not shown). As shown in figure 4, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT treatment induced a significant proliferation block in T-ALL cell lines RPMI 8402, KOPTKl and pancreatic cancer line PANCl. Similarly, 6-(4-Tert- Butylphenoxy)PyridinAmine (13) significantly inhibited the growth of nRas driven ma cell lines (Fig 4). However neither DAPT nor 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) had any effect on the eration ofNotch-independent RAJI cells (data not shown). 6-(4-Tert-Butylphenoxy)PyridinAmine (13) but not DAPT blocks Notch signalling in NICD overexpressing human T-ALL and breast cancer cell line. 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) can block NICD mediated activation of the Notch pathway (figure 1). To determine whether 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) can also induce a proliferation block in NICD overexpressing cells, the human T- ALL cell line DND4l (DND4l-Parental) was transduced with a NICD sing irus to generate DND4l-NICD cell line. These two cell lines were treated with DMSO, 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) and DAPT. The treatment of DND4l-parental cell line with DAPT and 6-(4-Tert-Butylphenoxy)PyridinAmine (13) led to a downregulation of Hesl when compared to DMSO treated cells. However, when DND4l-NICD cells were treated with DMSO, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT, only 6-(4- Tert-Butylphenoxy)PyridinAmine (13), but not DAPT treatment caused a downregulation of Hesl (Figure 5A). In addition, these two cell lines were also monitored over several days for anti-proliferative effects of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) and DAPT. It was observed that while both ert—Butylphenoxy)PyridinAmine (I3) and DAPT treatment caused a cant proliferative block in the DND4l-Parental cell line e 5B), only 6-(4-Tert-Butylphenoxy)PyridinAmine (13) was able to induce a growth arrest in DND4l-NICD cells (Figure 5C). This data further strengthen the notion that the 6-(4-Tert- W0 2013/093885 46 Butylphenoxy)PyridinAmine (13) compound can block NICD mediated pathway activation and proliferation in human cancer cells.
To r strengthen the notion that 6-(4-Tert-Butylphenoxy)PyridinAmine (13) can block NICD mediated pathway activation and proliferation of human cancer cells, 7 human breast cancer cell lines was treated with this compound. HCC1187 cell line harbors a SECC22B-Notch2 chromosomal translocation, thus generating constitutively active form ofN2-ICD (figure 5D). Due to this on, HCC1187 cell lines do not respond to y- ase inhibitors such as DAPT. As shown is figure 5E, while DAPT treatment did not inhibit eration of HCCl 187 cell lines, 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment significantly induced a proliferation block in these cell line. 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) induces G0/G1 cell cycle arrest and apoptosis in human T cell acute lymphoblastic leukemia cell lines: As shown in figure 4 and 5, 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment ofhuman leukemic cell lines and human breast cancer cell lines negatively te proliferation. This 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated proliferative arrest could be due to induction of apoptosis or cell cycle arrest during different phases of cell cycle. In addition, inhibition ofNotch signalling using y-secretase inhibitors has been shown to induce GO/Gl cell cycle arrest in human TALL cell lines. Therefore to further elucidate the mechanisms responsible for 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) ed proliferative arrest cell cycle and apoptosis analyses were carried out. Human TALL cell lines (RPMI8402, KOPTKl, TALL1, CUTL1 and HPB ALL) and human breast cancer cell line HCC1187 were d with 6-(4-Tert-Butylphenoxy)PyridinAmine (I3)or DMSO for 2 days or 7 days. To investigate cell death, Annexin V ng was performed and proportion of apoptotic (AnnexinV positive) cell population was determined by flow cytometry analyses after 7 days of treatment. As shown in figure 6A, 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment induces significant apoptosis in RPMI8402, CUTL1, KOPTKl, TALL1 and HPB ALL . Similarly, 6-(4-Tert-Butylphenoxy)PyridinAmine induces apoptosis in human breast cancer cell line HCC1187 (figure 6C).
In addition to induction of apoptosis, the proliferative arrest observed in 6-(4-Tert- henoxy)PyridinAmine (13) treated human leukemic cell lines and breast cancer cell line also s to be due to cell cycle arrest in GO/Gl phase of the cell cycle. Leukemic cell lines (RPMI8402, KOPTKl and TALL1) and breast cancer cell lines were treated with 6-(4- Tert-Butylphenoxy)PyridinAmine (13) for 48 hours and cell cycle status was determined using Ki67 and Hoechst stain. As shown in figure 6B and 6D, 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) induces an arrest in the G0/Gl phase of the cell cycle, a phenotype normally observed due to inhibition ofNotch signalling . 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated Notch signalling inhibition induces C2C 12 myoblast differentiation: To r confirm the Notch inhibitory potential of 6-(4-Tert-Butylphenoxy)Pyridin- 3-Amine (13) in different systems, C2C12 myoblast differentiation was used as a filnctional assay. The Notch pathway tion in C2C12 myoblasts retains them in an undifferentiated state, while abrogation ofNotch signalling induces their differentiation. C2C 12 myoblasts were treated with DMSO, 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) and DAPT and grown to 100% confluency for 3 days. After three days, cells were fixed and stained with antibodies against Myosin Heavy Chain (MHC) protein. Cell nuclei were counterstained with DAPI. C2C12 myoblasts grown in the presence of 10% serum (growth medium) maintain their undifferentiated state, while the cells treated with DAPT and 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) started to differentiate into multinucleated MHC positive myotubes (data not shown). 6-(4-Tert-Butylphenoxy)PyridinAmine (13) does not impede upon Wnt and og signalling cascades One of the ns about the activity of the chemical compound ert- Butylphenoxy)PyridinAmine (I3) is its city towards the Notch signalling y.
In order to test whether 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) could also block other developmental pathways, Applicants have tested its ability to block the Wnt and Hedgehog signalling pathways. In summary, to measure Wnt signalling HeLa cells were transfected with a plasmid containing a promoter consisting of TCF/LEF binding sites and thereby driving the sion of a luciferase gene (TOP-luciferase). To activate the Wnt pathway, a plasmid encoding for B-catenin was co-transfected into HeLa cells. The co-transfected cells were incubated in the presence or e of the 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) chemical nd. Transient introduction of B-catenin leads to an lation of Wnt signalling as measured by nin—TCF/LEF driven luciferase activity. Importantly, 6-(4- Tert-Butylphenoxy)PyridinAmine (13) treatment of cells with ted Wnt signalling does not block the Wnt pathway activation (data not shown).
W0 2013/093885 48 Using a similar strategy, Hedgehog signalling was activated in HeLa cells by introducing the Glil transcription factor and pathway tion was monitored using a promoter sequence containing Glil binding sites driving the luciferase sion. The treatment of these cells with 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) did not inhibit the Hedgehog signalling cascade (data not . Taken together these data suggest that the 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) chemical nd may not impair other developmental pathways and might be specific for Notch signalling inhibition. However it still needs to be determined whether the resistance of Wnt and og signalling s 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) is cell type specific or r it is a general phenomenon.
In vivo effects of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) in C57BL6 mice: Notch signalling regulates homeostasis of several organs during development. For example, Notchl mediated pathway activation is essential for T cell development in the thymus (Radtke et al., 1999). However, the Notchl driven T cell development does not appear to be dependent on MAMLl, as the loss ofMAMLl did not perturb T cell development in the mice. This could be due to a compensatory mechanism by MAML2 and MAML3 family members for the loss of MAMLl. In the spleen, Notch2 driven signalling exclusively via MAMLl is required for MZB cell development. Genetic ablation loss ofNotch2 and MAMLl cause a block in the pment ofMZB cells (Wu et al., 2007, Saito et al., 2003, ). In addition, Notch signalling via both Notchl and Notch2 is essential for the maintenance of the crypt compartment. A compound genetic ablation ofNotchl and Notch2 in the intestine leads to goblet cell metaplasia. Applicants therefore, investigated whether 6-(4-Tert- Butylphenoxy)PyridinAmine (13) could impair above-mentioned Notch-dependent developmental processes.
In in vitro culture assays, chemical compound 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) was able to block Notchl and Notch2 mediated pathway activation. ore, Applicants hypothesized that treatment of mice with 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) may lead to a goblet cell metaplasia of the intestine. To test this hypothesis, mice were intra peritoneally (i.p) ed with 25 mg/kg of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) for 7 consecutive days. On day 8, animals were sacrificed and intestinal s were fixed and embedded in paraffin. Histological analyses were carried out using Alcian blue to stain for W0 2013/093885 49 goblet cells. singly, despite its y to block both Notchl and Notch2 mediated pathway activation in in vitro cultures, the intestinal tissue of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treated mice was completely normal with intact architecture and no indication of goblet cell metaplasia (data not shown). Similarly, the effect of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) on body weight changes was also monitored. Mice were injected for 5 consecutive days with 25 mg/kg of 6-(4-Tert- Butylphenoxy)PyridinAmine (I3) and the changes in body mass were recorded. The treatment of mice with 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) did not cause a loss in the body weight. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) ent induces a block in MZB cell development: ants hypothesized that 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated inhibition ofNotch2 signalling should lead to a block in MZB cell development in the spleen.
MZB cell development was assessed by flow cytometry staining of splenocytes with antibodies directed against B220, CD21 and CD23. As shown in figure 7B treatment of mice with 6-(4-Tert-Butylphenoxy)PyridinAmine (13) leads to a reduction in the percentage of MZB cell population in the spleen. In addition, the loss ofMZB cell population in the spleen also s in the absolute number ofMZB cells (figure 7C).
Therefore, 6-(4-Tert-Butylphenoxy)PyridinAmine (I3) mediated block in MZB cell development mimics loss ofNotch2 and MAMLl phenotype. However, it is still need to be seen, whether 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) exert its Notch inhibitory effect only via MAMLl or it could block Notch signalling via other MAML family members as well. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treatment slows tumor growth of human T cell leukemia in a xenotransplantation model: Activation ofNotch signalling due to activating mutations in different components of the pathway are known to cause more than 50% of the human T cell acute lymphoblastic leukemias. Therefore, Applicants d to investigate the anti-cancer ty of the al compound ert-Butylphenoxy)Pyridin—3-Amine (13) in Notch driven human T cell leukemia in vivo. To achieve this goal, xenotransplant models of human leukemia were ished using NOD/SCID yc'/' mice. Human T-ALL cell lines HPB ALL and RPMI 8402 were used for this purpose. The HPB ALL cell line harbours a L1575P mutation in the heterodimerization domain and an insertion in the PEST domain of the Notchl receptor, thereby constitutively activating the Notchl signalling . rly, RPMI 8402 cells exhibit ligand independent Notch ling activation due to an ion at 1584 a.a residue in the heterodimerization domain and also an inactivating mutation (R465H) in the E3 ligase FBW7.
Both these cells line were found to respond to 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (13) treatment in in vitro culture assays in terms of proliferation and/or downregulation of the Notch target genes. To determine whether these cell lines establish leukemia in a xenotransplant setting, one million cells from each line were intra venously (i.v) injected into NOD/SCIDyc'/' mice. The animals developed leukemia with 100% ance and die within 4 weeks after transplantation.
Once RPMI 8402 and HPB ALL cell lines were shown to p ia in a ansplantation assay, they were transduced with a lentivirus constitutively expressing luciferase gene. This allowed Applicants to visualize and monitor the leukemia progression in the mice using the Caliper IVIS en) live imaging detection system. In order to determine the anti-cancer efficacy of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) in established tumors, a maintenance experiment was performed. One million HPB ALL cells were injected (iv) into NOD/SCID yc'/' mice. Mice were monitored for leukemia development by detecting luciferase sing ic cells. Once the disease was established around day 15, the mice were split into two groups. One group was treated with oil as a control and the second group was treated with 25 mg/kg of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) on a daily basis. As shown in figure 8A, the mice treated with oil p leukemia with 100% penetrance while leukemia in the 6-(4-Tert- Butylphenoxy)PyridinAmine (13) treated mice did not progress at the same rate as in the oil treated group (Figure 8A).
Similarly in a preliminary experiment, NOD/SCID yc'/' mice were transplanted with 5 x 105 RPMI 8402 cells and treated with oil or 6-(4-Tert-Butylphenoxy)PyridinAmine (13) following the establishment of the disease. As shown in figure 8B, animals treated with oil, developed leukemia while 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treated mice were free of the disease. Furthermore, histological analyses revealed that in oil treated mice, ic cells progressed to infiltrate the liver, but 6-(4-Tert-Butylphenoxy)PyridinAmine (13) treated mice did not develop any metastatic lesions in the liver (data not shown). Since these animals were treated with the chemical compound 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) for 27 days, the intestinal tissue was analyzed to detect any toxicity in the gut.
Alcian blue ng of intestinal tissue did not reveal any abnormality in the goblet cell numbers and intestinal ecture (data not shown).
Taken together Applicants’ data from xenotransplantation model for human leukemia suggest that ert-Butylphenoxy)Pyridin—3-Amine (13) has the ability to slow down disease progression of an already established ia. Because of its ability to impact tumor progression, 13 may be a suitable candidate for further development as an anti-cancer agent.
MMTV-ErbB2 mouse mammary tumors exhibit Notch signalling activation and effect of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) on mammary tumor progression.
In human breast cancer, high levels ofNotchl and Jaggedl proteins correlated with a poor survival of breast cancer patients. In addition, activation ofNotch signalling in human breast cancer also facilitates bone and lung metastasis. Therefore in order to determine anti- cancer potential of chemical compound 6-(4-Tert-Butylphenoxy)PyridinAmine (13) in breast cancer, a mouse model of breast cancer was investigated. The mouse mammary tumor virus (MMTV) driven overexpression of ErbB2 is known to cause mouse mammary .
MMTV-ErbB2 transgenic mice develop y tumors with a latency of about 5-6 months along with the development of lung metastasis. One of the characteristics of MMTV-ErbB2 mouse mammary tumors is the presence of predominantly l epithelial cell types. Notch signalling is known to drive luminal cell differentiation from mouse mammary stem cells.
Therefore Applicants hypothesized that the activation of the Notch pathway in rbB2 mammary tumors may contribute towards tumorigenesis in part by favouring l epithelial cell differentiation. To this end, Applicants investigated the levels ofNotch signalling activation in rbB2-IRES-Cre mammary tumors by measuring the levels of Hesl by Western blotting. As shown in figure 9A, MMTV-ErbB2 driven mammary tumors s very high levels of Hesl protein compared to age matched normal mammary glands.
To igate the effect of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) on breast cancer development, MMTV-ErbB2 mammary tumors were harvested from FVB mice carrying the MMTV-ErbB2 transgene. A single cell suspension was prepared and 5x105 tumor cells were injected into an empty fat pad of a recipient FVB mouse. Once palpable tumors had developed, recipient mice were treated with either oil or 25 mg/kg of 6-(4-Tert- Butylphenoxy)PyridinAmine (13) on alternate days until the end of the experiment. The tumor volume was ed and recorded on regular intervals. Preliminary results showed that the treatment of tumor bearing recipient mice with 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) caused significant tumor growth retardation when compared to mice d with oil alone (Figure 9B). This data showed that 6-(4-Tert-Butylphenoxy)PyridinAmine (13) has the ability to slow down the growth of established breast cancer. 6-(4-Tert-Butylphenoxy)PyridinAmine (13) blocks Notch ling in primary human T cell acute lymphoblastic leukemias: To further investigate the Notch inhibitory effect of 6-(4-Tert-Butylphenoxy)Pyridin- 3-Amine (13) in a relevant pathological condition, primary human TALL samples were d for the activation of Notch signalling . An accumulation of active form of Notch (N1CD) was used as a biomarker for pathway activation. Several primary human TALL exhibited an accumulation of nic NICD and ent of these tumors with 6-(4-Tert- Butylphenoxy)PyridinAmine (13) leads to a downregulation of this protein. Moreover, the downregulation ofNICD in these primary human TALL samples correlates with a proliferative arrest (data not shown). On the contrary, primary human TALL samples that do not show detectable levels ofNICD, did not respond to 6-(4-Tert-Butylphenoxy)Pyridin Amine (13) treatment (data not .
These data indicates that an accumulation ofNICD can be used as a biomarker for Notch pathway activation and predict treatment outcome using Notch inhibitor 6-(4-Tert- Butylphenoxy)PyridinAmine (13).
Different derivatives of 6-(4-Tert-Butylphenoxy)PyridinAmine (13) exhibit an ability to block Notch signalling activation in DL4-N1 coculture assay: In order to enhance the Notch inhibitory activity as well as efficacy of parental 6-(4- utylphenoxy)PyridinAmine (13) compound, different chemical derivatives of 13 were tested in DL4-Nl coculture assay. Screening of more than 40 different chemical derivatives of 6-(4-Tert-Butylphenoxy)Pyridin—3-Amine (I3) yielded following compounds for their ability to block Notch y activation in coculture assay (figure 10).
I3-A). 6-(4-cyclohexylphenoxy)pyridin—3-amine I3-B) 6-(4-(tert-Pentyl)phenoxy)pyridin—3-amine (CAS # 10365331) I3-C) tert-butyl)phenoxy)aniline (CAS # 567056) I3-D). 6-(4-Butylphenoxy)pyridinamine I3-E). 4-(4-(tert-pentyl)phenoxy)aniline (CAS # 3280321) I3-F). 4-(4-cyclohexylphenoxy)aniline (CAS # 706823) 13 -G) 6-(4-((3r,5r,7r)-adamantan— l -yl)phenoxy)pyridinamine I3-H). 6-(3-(tert-butyl)phenoxy)pyridin—3-amine (CAS # 10983668) 13-1). 4-(4-(tert-butyl)phenoxy)fluoroaniline (CAS # 9467859) I3-J). 4-(4-isopropylphenoxy)aniline I3-K). 6-(4-(2,4,4-trimethylpentanyl)phenoxy)pyridinamine I3-L). 4-(4-cyclohexylphenoxy)fluoroaniline I3-M). o(4-(tert—pentyl)phenoxy)aniline I3-N). 6-(4-(2-methylpentanyl)phenoxy)pyridinamine 13 -O). 4-(4-((3r,5r,7r)-adamantan— l -yl)phenoxy)aniline 13 -P). 4-(4-((3r,5r,7r)-adamantan— l -yl)phenoxy)-3 -fluoroaniline As shown in figure 10, some of these derivatives (I3-A, I3-B, I3-C, I3-E, I3-G, I3-H, I3-M and I3-N) block Notch signalling to comparable levels to al nd 13, while derivatives I3-F and 13-1 appears to have enhanced activity Example 3 Chemical synthesis of the Derivative and Precusors Thereof 4-(2-methylpentanyl)phen0l 4-Butyrylphenol (1000 mg, 6.09 mmol, 1.00 eq) was suspended in toluene (25 mL) and DCM (5mL) and cooled to 0°C. 2M MCgAl solution in toluene (7 mL, 14.01 mmol, 2.30 eq) was added dropwise whereby the starting material was dissolved. After stirring at room temperature for 15 h, the reaction e was again cooled to 0°C and TMSOSOzCFg (1.1 mL, 6.09 mmol, 1.00 eq) was added dropwise. After stirring at room temperature for 3 d, the reaction was quenched by pouring the mixture into ice-water. After ication with 40% H3PO4, the product was extracted with ethyl acetate (3x) and the organic layers were washed with H3PO4-acidic sat. aq. NaCl on. The solvent was removed under reduced pressure at oC. The ing crude product was purified by flash column chromatography (SiOz; DCM/petrolether 1:1 to 2:1) to give the title compound as colourless oil (188 mg, with a purity of around 90% (by NMR), 0.95 mmol, 15 % yield). Rf = 0.60 (DCM/MeOH 4%).
HRMS (ESI) calcd. for O' [M-H]' 177.1279, found: 177.1284. 1H NMR (400 MHz, CDClg) 5 7.24 — 7.18 (m, 2H, aromatic H), 6.82 — 6.76 (m, 2H, aromatic H), 5.12 (s, 1H, OH), 1.60 — 1.52 (m, 2H, 2CH2CH2CH3), 1.27 (s, 6H, C(CH3)2CH2CH2CH3), 1.16 — 1.01 (m, 2H, C(CH3)2CH2CH2CH3), 0.83 (t, .1: 7.3 Hz, 3H, C(CH3)2CH2CH2CH3). 13C NMR (101 MHz, CDC13)8 , 142.29, 127.11, 114.85, 47.35, 37.25, 29.23, 18.09, 14.90.
General Procedure A: The respective nitropyridines or nitrobenzenes and the particular s were dissolved in DMF or DMSO. Anhydrous K2C03 was added and the reaction mixture was stirred at room ature, unless otherwise , until complete conversion. The reaction was then quenched by the addition of H20 and the product was extracted with EtOAc or Et20. The organic layers were washed with 1M aq. NaOH solution (1x) and ards with sat. aq.
NaCl solution (1x). The solvent was removed to dryness under reduced pressure at 30°C. The residue was resolved in DCM and filtered through cotton to remove inorganic salts. The crude t was purified by flash column chromatography to afford the corresponding title compounds (I3-n, I3-nA to I3-nP). 2-(4-(tert-butyl)phenoxy)nitr0pyridine, I3-n ing procedure A, 2-chloronitropyridine (501 mg, 3.16 mmol, 1.00 eq) and 4-tert— butylphenol (611 mg, 4.07 mmol, 1.29 eq) were dissolved in DMF (6.0 mL). Anhydrous K2C03 (654 mg, 4.73 mmol, 1.50 eq) was added and the reaction mixture was stirred at room temperature for 14 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100 to 1:50) to afford the title compound as colourless solid (820 mg, 3.01 mmol, 95% yield). Rf: 0.40 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C15H17N203+ [M+H]+ 273.1234, found: 273.1229.1H NMR (400 MHz,CDC13)5 9.06 (d, J: 2.8 Hz, 1H, aromatic H), 8.46 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.53 —7.42 (m, 2H, aromatic H), 7.17 — 7.05 (m, 2H, aromatic H), 7.01 (d, J: 9.1 Hz, 1H, aromatic H), 1.35 (s, 9H, C(CH3)3). 13C NMR (101 MHz, CDC13)8 167.20, 150.49, 148.98, , 140.29, 134.95, 126.99, 120.84, 111.38, 34.72, 31.57.
W0 2013/093885 55 ycl0hexylphenoxy)nitr0pyridine, I3-nA Following procedure A, 2-chloronitropyridine (300 mg, 1.89 mmol, 1.00 eq) and 4- cyclohexylphenol (417 mg, 2.37 mmol, 1.25 eq) were dissolved in DMSO (6 mL).
Anhydrous K2C03 (397 mg, 2.87 mmol, 1.52 eq) was added and the reaction mixture was stirred at room temperature for 27 h. After extraction with EtzO, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100 to 1:75) to afford the title compound as colourless solid (600 mg, 2.01 mmol, quant. yield). Rf: 0.35 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C17H19N203+ [M+H]+ 299.1390, found: 299.1392. 1H NMR (400 MHz, CDC13)8 9.06 (d, J: 2.9 Hz, 1H, aromatic H), 8.46 (dd, J: 9.1, 2.9 Hz, 1H, ic H), 7.31 — 7.22 (m, 2H, aromatic H), 7.07 (dd, J: 8.7, 2.3 Hz, 2H, aromatic H), 7.00 (d, J: 9.1 Hz, 1H, aromatic H), 2.58 — 2.51(m, 1H, cyclohexyl H), 2.01 — 1.64 (m, 5H, cyclohexyl H), 1.56 — 1.10 (m, 5H, cyclohexyl H). 13C NMR (101 MHz, CDC13)8 167.18, 150.71, 145.88, 145.17, 140.22, 134.89, 128.30, 121.12, 111.30, 44.08, 34.59, 26.95, 26.19. -nitr0(4-(tert-pentyl)phenoxy)pyridine, I3-nB Following procedure A, 2-chloronitropyridine (303 mg, 1.91 mmol, 1.00 eq) and 4-tert— pentylphenol (397 mg, 2.42 mmol, 1.27 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (403 mg, 2.92 mmol, 1.53 eq) was added and the reaction e was stirred at room ature for 7 h. After extraction with Et20, the crude product was d by flash column chromatography (Si02; EtOAc/petrolether 1:100) to afford the title compound as colourless solid (530 mg, 1.85 mmol, 97% yield). Rf: 0.31 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C16H19N203+ [M+H]+ 287.1390, found: 287.1381. 1H NMR (400 MHz,CDC13)5 9.07 — 9.05 (m, 1H, aromatic H), 8.45 (dd, J: 9.4, 2.4 Hz, 1H, ic H), 7.40 (d, J: 8.6 Hz, 2H, aromatic H), 7.09 (d, J: 8.6 Hz, 2H, aromatic H), 6.99 (d, J: 9.2 Hz, 1H, ic H), 1.67 (q, .1 = 7.4 Hz, 2H, Ar-C(CH3)2CH2CH3), 1.31 (s, 6H, Ar-C(CH3)2CH2CH3), 0.73 (t, .1: 7.4 Hz, 3H, Ar-C(CH3)2CH2CH3). 13c NMR (101 MHz, CDClg) 5 167.17, 150.46, , 145.20, 140.27, 134.90, 127.56, 120.72, 111.28, 37.89, 37.06, 28.55, 9.26.
I-(tert-butyl)(4-nitr0phen0xy)benzene, I3-nC +011...
Following procedure A, 4-fluoronitrobenzene (500 mg, 3.54 mmol, 1.00 eq) and 4-tert— henol (671 mg, 4.46 mmol, 1.26 eq) were dissolved in DMF (6.0 mL). Anhydrous K2C03 (857 mg, 6.20 mmol, 1.75 eq) was added and the reaction mixture was stirred at room temperature for 52 h. After extraction with EtOAc, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100) to afford the title compound as pale yellow solid (860 mg, 3.17 mmol, 89 % yield). Rf: 0.72 /PE 1:9). HRMS (ESI) calcd. for C16H18N03+ [M+H]+ 272.1281, found: 272.1272. 1H NMR (400 MHz,CDC13) 8 8.24 — 8.16 (m, 2H, aromatic H), 7.49 — 7.39 (m, 2H, aromatic H), 7.06 — 6.97 (m, 4H, aromatic H), 1.35 (s, 9H, C(CH3)3). 13C NMR (101 MHz, CDC13)8 163.82, 152.29, 148.59, 142.54, 127.27, 126.03, 120.15, 116.98, 34.67, 31.58. 2-(4-butylphenoxy)nitr0pyridine, I3-nD Following procedure A, 2-chloronitropyridine (103 mg, 0.65 mmol, 1.00 eq) and 4- butylphenol (126 mg, 0.84 mmol, 1.29 eq) were dissolved in DMF (2.5 mL). Anhydrous K2C03 (143 mg, 1.04 mmol, 1.59 eq) was added and the reaction mixture was stirred at room temperature for 6 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100) to afford the title nd as colourless solid (190 mg, 0.70 mmol, quant. yield). Rf: 0.33 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C15H17N203+ [M+H]+ 273.1234, found: 26.1H NMR (400 MHz,CDC13)8 9.05 (dd, J: 2.9, 0.6 Hz, 1H, ic H), 8.46 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.32 — 7.21 (m, 2H, aromatic H), 7.11 — 7.02 (m, 2H, aromatic H), 7.00 (dd, J: 9.0, 0.6 Hz, 1H, aromatic H), 2.69 — 2.60 (m, 2H, Ar-CH2CH2CH2CH3), 1.70 — 1.57 (m, 2H, Ar- CH2CH3), 1.39 (dq, J: 14.6, 7.3 Hz, 2H, Ar-CH2CH2CH2CH3), 0.95 (t, J: 7.3 Hz, 3H, Ar-CH2CH2CH2CH3). 13c NMR (101 MHz,CDC13)8 , 150.75, , 140.86, 140.30, 134.92, 129.91, 121.22, 111.32, 35.21, 33.67, 22.51, 14.08.
W0 2013/093885 57 I-nitr0(4-(tert-pentprhen0xy)benzene, I3-nE @001.
Following procedure A, 4-fluoronitrobenzene (318 mg, 2.25 mmol, 1.00 eq) and — pentylphenol (460 mg, 2.28 mmol, 1.24 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (465 mg, 3.37 mmol, 1.49 eq) was added and the reaction mixture was stirred at room temperature for 2 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100) to afford the title compound as colourless solid (533 mg, 1.87 mmol, 83% yield). Rf: 0.70 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C17H20N03+ [M+H]+ 285.1365, found: 285.1359.1H NMR (400 C13)5 8.25 — 8.14 (m, 2H, aromatic H), 7.44 — 7.32 (m, 2H, aromatic H), 7.06 — 6.96 (m, 4H, aromatic H), 1.66 (q, .1: 7.4 Hz, 2H, Ar-C(CH3)2CH2CH3), 1.31 (s, 6H, Ar- C(CH3)2CH2CH3), 0.71 (t, .1: 7.4 Hz, 3H, Ar-C(CH3)2CH2CH3). 13c NMR (101 MHz, CDC13)8 163.81, 152.24, 146.94, 142.56, 127.91, , 120.06, 116.99, 37.89, 37.06, 28.63, 9.27.
I-cyclohexyl—4—(4-nitr0phen0xy)benzene, I3-nF Following procedure A, 4-fluoronitrobenzene (325 mg, 2.30 mmol, 1.00 eq) and 4- cyclohexylphenol (519 mg, 2.94 mmol, 1.28 eq) were dissolved in DMSO (6 mL).
Anhydrous K2C03 (513 mg, 3.72 mmol, 1.61 eq) was added and the on mixture was stirred at room ature for 48 h. After extraction with EtzO, the crude product was purified by flash column chromatography (Si02; petrolether 1:100 to 1:50) to afford the title compound as pale yellow solid (640 mg, 2.15 mmol, 93% yield). Rf: 0.55 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C18H20N03+ [M+H]+ 298.1438, found: 298.1442. 1H NMR (400 MHz, CDC13)8 8.31 — 8.11 (m, 2H, aromatic H), 7.26 (d, J: 2.3 Hz, 2H, aromatic H), 7.13 — 6.92 (m, 4H, aromatic H), 2.57 — 2.50 (m, 1H, cyclohexyl H), 2.09 — 1.67 (m, 5H, cyclohexyl H), 1.59 — 1.18 (m, 5H, cyclohexyl H). 13c NMR (101 MHz,CDC13) 5 163.89, 152.61, 145.58, , , 126.04, 120.49, 116.99, 44.14, 34.72, 26.99, 26.23. 2-(4-((3r,5r, 7r)-adamantan-I-yl)phen0xy)nitr0pyridine, I3-nG Following procedure A, 2-chloronitropyridine (300 mg, 1.89 mmol, 1.00 eq) and 4- adamantylphenol (546 mg, 2.39 mmol, 1.26 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (662 mg, 4.79 mmol, 2.53 eq) was added and the reaction e was d at room temperature for 42 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100 to 1:50) to afford the title compound as colourless solid (664 mg, 1.89 mmol, quant. yield). Rf: 0.36 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C21H23N203+ [M+H]+ 351.1703, found: 351.1701.1H NMR (400 MHz, CDC13)5 9.06 (d, J: 2.8 Hz, 1H, aromatic H), 8.45 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.52 — 7.39 (m, 2H, aromatic H), 7.16 — 7.06 (m, 2H, aromatic H), 7.00 (d, J: 9.1 Hz, 1H, aromatic H), 2.13 — 2.10 (m, 3H, tyl H), 1.94 (d, J: 3.0 Hz, 5H, adamantyl H), 1.87 — 1.70 (m, 5H, tyl H). 13c NMR (101 MHz,CDC13)8 167.19, 150.51, 149.18, 145.20, 140.26, 134.89, 126.53, , 111.32, 43.35, 36.82, 36.18, 29.02. 2-(3-(tert-butyl)phenoxy)nitr0pyridine, I3-nH omN / Following procedure A, 2-chloronitropyridine (300 mg, 1.89 mmol, 1.00 eq) and 3-tert— butylphenol (358 mg, 2.38 mmol, 1.26 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (420 mg, 3.04 mmol, 1.60 eq) was added and the reaction mixture was stirred at room temperature for 70 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:100) to afford the title compound as colourless solid (512 mg, 1.88 mmol, 99% . Rf: 0.37 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C15H17N203+ [M+H]+ 273.1234, found: 273.1232.1H NMR (400 MHz,CDC13)8 9.08 — 9.04 (m, 2H, aromatic H), 8.47 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.39 (t, J: 7.9 Hz, 1H, ic H), 7.33 (ddd, J: 7.9, 1.8, 1.2 Hz, 1H, aromatic H), 7.17 (t, J: 2.1 Hz, 1H, aromatic H), 7.01 (dd, J: 9.1, 0.5 Hz, 1H, aromatic H), 6.98 (ddd, J: 7.9, 2.4, 1.2 Hz, 1H, aromatic H), 1.34 (s, 9H, C(CH3)3). 13c NMR (101 MHz, CDC13) 5 167.21, 153.88, 152.76, 145.26, 140.31, 134.93, 129.50, 123.17, 118.62, 118.47, 111.28, 35.02, 31.36.
I-(4-(tert-butyl)phen0xy)flu0r0nitr0benzene, I3-nI tow... ing procedure A, 3,4-difluoronitrobenzene (401 mg, 2.52 mmol, 1.00 eq) and 4-tert— butylphenol (477 mg, 3.18 mmol, 1.26 eq) were dissolved in DMSO (6 mL). ous K2C03 (522 mg, 3.78 mmol, 1.50 eq) was added and the on mixture was stirred at room temperature for 19 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:50) to afford the title compound as colourless oil (722 mg, 2.52 mmol, 99% yield). Rf: 0.54 /PE 1:20). HRMS (ESI) calcd. for C16H17FN03+ [M+H]+ 290.1187, found: 290.1194. 1H NMR (400 MHz,CDC13)5 8.07 (dd, J: 10.3, 2.7 Hz, 1H, aromatic H), 7.96 (ddd, J: 9.1, 2.7, 1.5 Hz, 1H, aromatic H), 7.49 — 7.38 (m, 2H, aromatic H), 7.09 — 6.99 (m, 2H, aromatic H), 6.96 (dd, J: 9.1, 8.0 Hz, 1H, aromatic H), 1.35 (s, 9H, C(CH3)3). 13c NMR (101 MHz, CDC13) 5 153.35, 152.23, 151.93, 151.82, 150.84, 148.70, 142.40, , 127.29, 120.68, 120.64, 119.38, 117.73, 117.71, 113.28, 113.05, 34.65, 31.54.
I-(4-cyclohexylphenoxy)flu0r0nitr0benzene, I3-nJ 5500...
Following procedure A, 3,4-difluoronitrobenzene (509 mg, 3.20 mmol, 1.00 eq) and 4- exylphenol (691 mg, 3.93 mmol, 1.23 eq) were dissolved in DMSO (6 mL).
Anhydrous K2C03 (664 mg, 4.81 mmol, 1.50 eq) was added and the reaction mixture was stirred at room temperature for 23 h. After extraction with EtzO, the crude product was purified by flash column tography (Si02; EtOAc/petrolether 1:50) to afford the title compound as pale yellow solid (1002 mg, 3.18 mmol, 99% . Rf: 0.51 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C18H19FN03+ [M+H]+ 316.1343, found: 316.1348.1H NMR (400 MHz,CDC13)8 8.09 (dd, J: 10.3, 2.7 Hz, 1H, aromatic H), 7.97 (ddd, J: 9.1, 2.7, 1.4 W0 2013/093885 60 Hz, 1H, aromatic H), 7.33 — 7.22 (m, 2H, aromatic H), 7.08 — 6.99 (m, 2H, aromatic H), 6.96 (dd, J: 9.1, 8.0 Hz, 1H, aromatic H), 2.58 — 251 (m, 1H, cyclohexyl H), 1.98 — 1.73 (m, 5H, cyclohexyl H), 1.52 — 1.20 (m, 5H, cyclohexyl H). 13C NMR (101 MHz, CDClg) 5 153.35, 152.51, 152.01, 151.90, 150.84, 145.68, 142.39, 142.32, 128.67, 120.70, 120.66, 119.73, 117.70, 117.68, , 113.08, 44.09, 34.69, 26.97, 26.21. 2-flu0r0nitr0-I-(4-(tert-pentyl)phenoxy)benzene, I3-nK 00°61.
Following procedure A, 3,4-difluoronitrobenzene (502 mg, 3.16 mmol, 1.00 eq) and 4-tert— pentylphenol (693 mg, 4.22 mmol, 1.34 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (656 mg, 4.75 mmol, 1.50 eq) was added and the reaction mixture was stirred at room temperature for 22 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:50) to afford the title compound as pale yellow oil (943 mg, 3.11 mmol, 99% yield). Rf: 0.61 /PE 1:20). HRMS (ESI) calcd. for C17H19N03+ [M+H]+ 304.1343, found: 304.1332.1H NMR (400 MHz,CDC13)5 8.08 (dd, J: 10.3, 2.7 Hz, 1H, aromatic H), 7.96 (ddd, J: 9.1, 2.7, 1.5 Hz, 1H, ic H), 7.42 — 7.35 (m, 2H, aromatic H), 7.07 — 6.99 (m, 2H, ic H), 6.95 (dd, J: 9.1, 8.0 Hz, 1H, aromatic H), 1.66 (q, .1: 7.4 Hz, 2H, Ar-C(CH3)2CH2CH3), 1.31 (s, 6H, Ar- C(CH3)2CH2CH3), 0.71 (t, .1: 7.4 Hz, 3H, Ar-C(CH3)2CH2CH3). 13c NMR (101 MHz, CDC13)5153.40, 152.19, 151.98,151.87, 150.88, 147.11, 127.97, 120.71, 120.68, 119.34, , 117.71, 113.11, 37.92, 37.08, 28.64. 2-methylpentan-2—y0phenoxy)nitr0pyridine, I3-nL ing procedure A, 4-(2-methylpentanyl)phenol (52 mg, 0.29 mmol, 1.00 eq) and 2- chloronitropyridine (57 mg, 0.36 mmol, 1.23 eq) were dissolved in DMSO (6 mL).
Anhydrous K2C03 (66 mg, 0.48 mmol, 1.65 eq) was added and the reaction mixture was stirred at room temperature for 27 h. After extraction with EtzO, the crude product was purified by flash column chromatography (Si02; EtOAc/petrolether 1:50) to afford the title W0 2013/093885 61 compound as colourless solid (86 mg, 0.29 mmol, 98% yield). Rf: 0.50 (EtOAc/PE 1:10).
HRMS (ESI) calcd. for C17H21N203+ [M+H]+ 301.1547, found: 301.1545.1H NMR (400 MHZ, CDClg) 5 9.07 (d, J: 2.7 Hz, 1H, aromatic H), 8.46 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.45 — 7.35 (m, 2H, aromatic H), 7.14 — 7.05 (m, 2H, aromatic H), 6.99 (dd, J: 9.0, 0.6 Hz, 1H, aromatic H), 1.65 — 1.54 (m, 2H, C(CH3)2CH2CH2CH3), 1.32 (s, 6H, C(CH3)2CH2CH2CH3), 1.19 — 1.05 (m, 2H, 2CH2CH2CH3), 0.84 (t, J = 7.3 Hz, 3H, C(CH3)2CH2CH2CH3). 13C NMR (101 MHz, CDClg) 5 167.17, 150.43, , 145.21, 140.26, 134.90, 127.44, 120.72, 111.29, 47.27, 37.74, 29.07, 18.08, 14.87. (3r, 5r, 7r)-I-(4-(4-nitr0phen0xy)phenyDadamantane, I3-nM £13118 Following procedure A, 4-fluoronitrobenzene (303 mg, 2.15 mmol, 1.00 eq) and 4- adamantylphenol (600 mg, 2.63 mmol, 1.22 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (450 mg, 3.26 mmol, 1.52 eq) was added and the reaction mixture was stirred at room temperature for 20 h. After extraction with Et20, the crude product was purified by flash column chromatography (Si02; petrolether 1:100 to 1:50) to afford the title compound as pale yellow solid (736 mg, 2.11 mmol, 98% yield). Rf: 0.73 (EtOAc/PE 1:10). HRMS (ESI) calcd. for C22H24N03+ [M+H]+ 350.1751, found: 350.1760.1H NMR (400 MHz, CDClg) 5 8.27 8.10 (m, 2H, aromatic H), 7.46 — 7.35 (m, 2H, aromatic H), 7.08 — 6.95 (m, 4H, aromatic H), 2.13 — 2.10 (m, 3H, adamantyl H), 1.93 (d, J: 2.9 Hz, 6H, adamantyl H), 1.88 — 1.70 (m, 6H, adamantyl H). 13C NMR (101 MHz, CDC13) 5 163.85, 152.30, 148.86, 142.53, 126.86, 126.85, , 120.18, 117.00, 43.40, 36.82, 36.17, 29.03. (3r, 5r, (4-(2-flu0r0nitr0phenoxy)phenyl)adamantane, I3-nN .
Following procedure A, fluoronitrobenzene (303 mg, 1.90 mmol, 1.00 eq) and 4- adamantylphenol (533 mg, 2.34 mmol, 1.23 eq) were dissolved in DMSO (6 mL). Anhydrous K2C03 (395 mg, 2.86 mmol, 1.50 eq) was added and the reaction mixture was stirred at room W0 2013/093885 62 temperature for 4 h. After extraction with EtZO, the crude product was purified by flash column tography (Si02; EtOAc/petrolether 1:100) to afford the title compound as colourless solid (703 mg, 1.91 mmol, quant. yield). Rf: 0.69 (EtOAc/PE 1:10). HRMS (APPI) calcd. for C22H22FNO+ [M]+ 367.1584, found: 367.1581.1H NMR (400 MHz, CDClg) 8 8.08 (dd, J: 10.3, 2.7 Hz, 1H, aromatic H), 7.96 (ddd, J: 9.1, 2.7, 1.5 Hz, 1H, ic H), 7.46 — 7.36 (m, 2H, aromatic H), 7.06 — 7.00 (m, 2H, aromatic H), 6.95 (dd, J: 9.1, 8.0 Hz, 1H, aromatic H), 2.17 — 2.07 (m, 3H, adamantyl H), 1.92 (d, J: 2.9 Hz, 5H, adamantyl H), 1.87 — 1.71 (m, 5H, adamantyl H). 13c NMR (101 MHz, CDC13) 5 153.36, 153.35, 152.24, 152.21, 151.99, 151.88, , 150.83, 148.98, 126.89, 120.70, 120.66, 119.44, 119.42, 117.72, 117.70, 113.30, 113.07, 43.38, 36.81, 36. 17, 29.02. 2-(4-is0pr0pylphen0xy)nitr0pyridine, I3-n0 Following procedure A, 2-chloronitropyridine (303 mg, 1.91 mmol, 1.00 eq) and 4-z's0- propylphenol (331 mg, 2.43 mmol, 1.27 eq) were dissolved in DMF (6.0 mL). Anhydrous K2C03 (398 mg, 2.88 mmol, 1.51 eq) was added and the reaction mixture was stirred at room temperature for 24 h. After extraction with Et20, the crude t was purified by flash column chromatography (Si02; petrolether 1:100) to afford the title compound as pale yellow solid (490 mg, 1.90 mmol, 99% . Rf: 0.40 (EtOAc/PE 1:20). HRMS (ESI) calcd. for C14H15N203+ [M+H]+ 259.1077, found: 259.1072.1H NMR (400 MHz,CDC13) 8 9.04 (d, J: 3.4 Hz, 1H, aromatic H), 8.44 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.37 — 7.28 (m, 2H, aromatic H), 7.12 — 7.06 (m, 2H, aromatic H), 7.03 — 6.97 (m, 1H, aromatic H), 2.96 (hept, .1: 6.9 Hz, 1H, )2), 1.30 (d, J: 7.0 Hz, 6H, CH(CH3)2). 13c NMR (101 MHz, CDCl3)5167.08, 150.67, 146.49, 145.02, 140.18, 134.81, 127.84, 121.11, 111.24, 33.62, 24.03. 0(4-(2,4,4-trimethylpentanyl)phen0xy)pyridine, I3-nP Following procedure A, 2-Chloronitropyridine (303 mg, 1.91 mmol, 1.00 eq) and 4-tert— octylphenol (495 mg, 2.40 mmol, 1.25 eq) were dissolved in DMSO (5 mL). Anhydrous W0 2013/093885 63 2012/057622 K2C03 (426 mg, 3.08 mmol, 1.61 eq) was added and the reaction mixture was d at 40°C for 28 h. After extraction with Et20, the crude product was purified by flash column chromatography (SiOz; EtOAc/petrolether 1:50) to afford the title nd as colourless solid (598 mg, 1.82 mmol, 95% . Rf: 0.65 (EtOAc/PE 1:10). HRMS (ESI) calcd. for C19H25N203+ [M+H]+ 329.1860, found: 329.1854.1H NMR (400 MHz,CDC13)8 9.07 (d, J: 2.8 Hz, 1H, aromatic H), 8.45 (dd, J: 9.1, 2.8 Hz, 1H, aromatic H), 7.52 — 7.40 (m, 2H, aromatic H), 7.14 — 7.03 (m, 2H, aromatic H), 6.97 (d, J: 9.1 Hz, 1H, aromatic H), 1.76 (s, 2H, Ar-C(CH3)2CH2C(CH3)3), 1.40 (s, 6H, Ar-C(CH3)2CH2C(CH3)3), 0.75 (s, 9H, Ar- C(CH3)2CH2C(CH3)3). 13C NMR (101 MHz, CDCl3) 8 167.24, 150.49, 148.12, 145.30, 140.29, 134.92, 127.78, 120.57, 111.15, 57.28, 38.63, 32.55, 31.93, 31.62.
General Procedure B: The respective nitro derivatives (I3-n, I3-nA to I3-nP) were first dissolved in MeOH or toluene, or directly added to a suspension of catalytic s of Pd (10%) on activated carbon powder in MeOH. The flask was purged with H2 (6x) and the reaction mixture was stirred at room temperature until complete conversion. The reaction mixture was then filtered h . The solvent was removed under reduced pressure at 30 oC and the crude product was purified by flash column chromatography to give the corresponding title nds (13, I3-A to I3-P). 6-(4-(tert-butyl)phen0xy)pyridinamine, I3 Following procedure B, I3-n (300 mg, 1.10 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (82 mg, 0.08 mmol Pd, 0.07 eq) in MeOH (15 mL). The flask was purged with H2 (6x) and the reaction mixture was stirred at room temperature for 2 h. The crude product was purified by flash column chromatography (SiO2; DCM/MeOH 1%) to give the title nd as pale beige solid (250 mg, 1.03 mmol, 94% yield). Rf: 0.40 (DCM/MeOH 4%). HRMS (ESI) calcd. for C15H19N20+ [M+H]+ 243.1492, found: 243.1487. 1H NMR (400 MHz, CDC13)5 7.69 (d, J: 3.0 Hz, 1H, aromatic H), 7.39 — 7.31 (m, 2H, aromatic H), 7.03 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.00 — 6.93 (m, 2H, aromatic H), 6.72 W0 2013/093885 64 (d, .1: 8.6 Hz, 1H, aromatic H), 3.48 (s, 1H, NH2), 1.31 (s, 9H, C(CH3)3). 13c NMR (101 MHz,CDC13)5156.62, , 146.33, 138.82, 134.06, 126.86, 126.47, , 112.36, 34.33, 31.52. 6-(4-cyclohexylphenoxy)pyridinamine, I3-A Following ure B, I3-nA (201 mg, 0.67 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (47 mg, 0.04 mmol Pd, 0.07 eq) in MeOH (15 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 5 h. The crude product was purified by flash column chromatography (Si02; DCM/MeOH 1%) to give the title compound as beige solid (190 mg, 0.71 mmol, quant. . Rf: 0.36 (DCM/MeOH 4%). HRMS (ESI) calcd. for C17H21N20+ [M+H]+ 269.1648, found: 269.1643. 1H NMR (400 MHz, CDClg) 8 7.70 (d, J: 2.9 Hz, 1H, aromatic H), 7.22 — 7.13 (m, 2H, aromatic H), 7.04 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.01 — 6.92 (m, 2H, aromatic H), 6.73 (dd, J: 8.8, 0.7 Hz, 1H, aromatic H), 3.36 (s, 2H, NH2), 2.51 —2.44 (m, 1H, cyclohexyl H), 1.97 — 1.67 (m, 5H, cyclohexyl H), 1.49 — 1.15 (m, 5H, cyclohexyl H). 13c NMR (101 MHz, CDC13)5156.88, 153.61, 143.47, 138.67, 134.21, 127.92, 126.96, 119.68, 112.38, 43.99, 34.68, 27.01, 26.25. 6-(4-(tert-pentprhenoxy)pyridin-3—amine, I3-B Following ure B, I3-nB (200 mg, 0.70 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (52 mg, 0.05 mmol Pd, 0.07 eq) in MeOH (15 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 3 h. The crude product was purified by flash column chromatography (Si02; DCM/MeOH 0.5%) to give the title compound as beige solid (107 mg, 0.42 mmol, 60% yield). Rf: 0.41 (DCM/MeOH 4%). HRMS (ESI) calcd. for N20+ [M+H]+ 257.1648, found: 257.1648. 1H NMR (400 MHz, CDClg) 5 7.73 (d, J: 2.9 Hz, 1H, aromatic H), 7.34 — 7.23 (m, 2H, aromatic H), 7.06 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.02 — 6.94 (m, 2H, aromatic H), 6.73 (d, J: 8.6 Hz, 1H, aromatic H), 3.35 (s, 2H, NH2), 1.62 (q, .1: 7.4 Hz, 2H, Ar- C(CH3)2CH2CH3), 1.27 (s, 6H, Ar-C(CH3)2CH2CH3), 0.70 (t, J = 7.4 Hz, 3H, Ar- 2CH2CH3). 13C NMR (101 MHz,CDC13)8 156.80, 153.36, 144.75, 138.72, 134.30, 127.19, 126.97, 119.13, 112.51, 37.63, 37.04, 28.64, 9.26. 4-(4-(tert-butyl)phen0xy)aniline, I3-C 00mm ing procedure B, I3-nC (300 mg, 1.11 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (59 mg, 0.06 mmol Pd, 0.05 eq) in MeOH (15 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 3.5 h. The crude product was purified by flash column chromatography (SiOz; ethyl acetate/petrolether 1:100 to 1:10) to give the title compound as dark yellow oil (244 mg, 1.01 mmol, 91% . Rf: 0.78 (DCM/MeOH 4%). HRMS (ESI) calcd. for C16H20NO+ [M+H]+ 242.1539, found: 242.1530.1H NMR (400 MHz,CDC13)8 7.33 — 7.27 (m, 2H, ic H), 6.91 — 6.84 (m, 4H, aromatic H), 6.72 — 6.66 (m, 2H, aromatic H), 3.49 (s, 2H, NH2), 1.31 (s, 9H, 3). 13C NMR (101 MHz,CDC13)8 156.55, 149.24, 145.05, 142.30, 126.45, 121.08, 116.90, 116.49, 34.33, 31.66. 6-(4-butylphenoxy)pyridinamine, I3-D Following procedure B, I3-nD (170 mg, 0.62 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (53 mg, 0.05 mmol Pd, 0.08 eq) in MeOH (15 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 1.5 h. The crude product was d by flash column chromatography (SiOz; DCM/MeOH 1%) to give the title compound as brown oil (133 mg, 0.55 mmol, 88% yield). Rf: 0.38 W0 2013/093885 66 2012/057622 (DCM/MeOH 4%). HRMS (ESI) calcd. for C15H19N20+ [M+H]+ 243.1492, found: 243.1485. 1H NMR (400 MHz, CDClg) 5 7.69 (d, J: 3.1 Hz, 1H, aromatic H), 7.20 — 7.09 (m, 2H, aromatic H), 7.03 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.00 — 6.91 (m, 2H, aromatic H), 6.72 (d, J: 8.6 Hz, 1H, aromatic H), 3.36 (s, 2H, NH2), 2.67 — 2.51 (m, 2H, Ar-CHZCHZCHZCHg), 1.66 — 1.52 (m, 2H, Ar-CHZCHZCHZCHg), 1.41 — 1.31 (m, 2H, Ar-CHZCHZCHZCHg), 0.93 (t, .1: 7.3 Hz, 3H, Ar-CHZCHzCHzCHg). 13C NMR (101 MHz, CDC13)8 156.83, 153.55, 138.75, 138.26, 134.12, 129.48, 126.90, 119.74, 112.28, 35.02, 33.74, 22.40, 14.02. 4-(4-(tert-pentyl)phen0xy)aniline , I3-E Following procedure B, I3-nE (313 mg, 1.10 mmol, 1.00 eq) was ved in MeOH (10 mL) and added to a suspension of Pd (10%) on activated carbon powder (52 mg, 0.05 mmol Pd, 0.04 eq) in MeOH (5 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 3 h. The crude product was purified by filtration through a thin Si02 layer (DCM/MeOH 4%) to give the title nd as beige oil (293 mg, 1.15 mmol, quant. yield). Rf: 0.09 /PE 1:20). HRMS (ESI) calcd. for C17H22N20+ [M+H]+ 256.1696, found: 256.1692.1H NMR (400 MHz, CDC13) 8 7.29 — 7.19 (m, 2H, aromatic H), 6.98 - 6.81 (m, 4H, aromatic H), 6.76 — 6.61 (m, 2H, aromatic H), 3.47 (s, 2H, NH2), 1.63 (q, .1 = 7.4 Hz, 2H, H3)2CH2CH3), 1.28 (s, 6H, Ar-C(CH3)2CH2CH3), 0.71 (t, J = 7.4 Hz, 3H, Ar-C(CH3)2CH2CH3). 13c NMR (101 MHz, CDClg) 5 156.45, 149.08, 143.29, 142.55, 127.08, 121.04, 116.79, 116.33, 37.50, 37.06, 28.69, 9.27. 4-(4-cycl0hexylphenoxy)aniline,I3-F Following procedure B, I3-nF (352 mg, 1.18 mmol, 1.00 eq) was dissolved in toluene (5 mL) and added to a sion of Pd (10%) on activated carbon powder (38 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (10 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 2 h. The crude product was purified by flash column chromatography (Si02; DCM) to give the title compound as beige solid (315 mg, 1.18 mmol, quant. yield). Rf: 0.86 (DCM/MeOH 4%). HRMS (ESI) calcd. for C18H22NO+ [M+H]+ 268.1696, found: 268.1692.1H NMR (400 MHz,CDC13)8 7.17 — 7.07 (m, 2H, aromatic H), 6.94 — 6.82 (m, 4H, aromatic H), 6.72 — 6.62 (m, 2H, aromatic H), 3.49 (s, 2H, NH2), 2.51 — 2.44 (m, 1H, cyclohexyl H), 1.98 — 1.68 (m, 5H, cyclohexyl H), 1.46 — 1.21 (m, 5H, cyclohexylH). 13C NMR (101 MHz,CDC13)5 , 149.13, 142.53, 142.05, 127.81, 121.02, , , 43.90, 34.79, 27.05, 26.27. 6-(4-((3r,5r, 7r)-adamantan-I-yl)phen0xy)pyridinamine, I3-G Following procedure B, I3-nG (278 mg, 0.79 mmol, 1.00 eq) was dissolved in toluene (10 mL) and added to a suspension of Pd (10%) on activated carbon powder (56 mg, 0.05 mmol Pd, 0.07 eq) in MeOH (10 mL). The flask was purged with H2 (3X) and the reaction mixture was stirred at room temperature for 3 h. The crude product was purified by flash column chromatography (SiOz; OH 0% to 1%) to give the title compound as colourless solid (176 mg, 0.55 mmol, 69% yield). Rf: 0.43 (DCM/MeOH 4%). HRMS (ESI) calcd. for N20+ [M+H]+ 321.1961, found: 321.1959.1H NMR (400 MHz,CDC13)8 7.71 (d, J: 3.0 Hz, 1H, aromatic H), 7.37 — 7.29 (m, 2H, aromatic H), 7.08 — 6.96 (m, 3H, aromatic H), 6.74 (d, J: 8.6 Hz, 1H, aromatic H), 3.40 (s, 2H, NH2), 2.12 — 2.09 (m, 3H, adamantyl H), 1.93 (dd, .1: 9.7, 3.0 Hz, 5H, adamantyl H), 1.86 — 1.70 (m, 5H, adamantyl H). 13c NMR (101MHz,CDC13)8156.73, 153.37, 151.35, 146.65, 138.76, , 128.14, 126.86, 126.06, 125.54, 124.88, 119.29, 112.40, 43.36, 43.22, 36.87, 36.84, 35.87, 29.02. 6-(3-(tert-butyl)phen0xy)pyridinamine, I3-H omN / W0 2013/093885 68 Following procedure B, I3-nH (362 mg, 1.33 mmol, 1.00 eq) was dissolved in MeOH (10 mL) and added to a suspension of Pd (10%) on activated carbon powder (44 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (5 mL). The flask was purged with H2 (5X) and the reaction mixture was stirred at room temperature for 2 h. The crude t was purified by flash column chromatography (SiOz; DCM/MeOH 0% to 1%) to give the title compound as pale brown oil (303 mg, 1.25 mmol, 94 % yield). Rf: 0.44 (DCM/MeOH 4%). HRMS (ESI) calcd. for C15H19N20+ [M+H]+ 243.1492, found: 243.1493.1H NMR (400 MHz,CDC13)5 7.92 (d, J: 3.0 Hz, 1H, aromatic H), 7.48 — 7.40 (m, 1H, aromatic H), 7.32 (ddd, J: 7.8, 1.9, 1.0 Hz, 1H, aromatic H), 7.29 — 7.27 (m, 1H, aromatic H), 7.24 (d, J: 3.0 Hz, 1H, aromatic H), 7.01 (ddd, J: 8.0, 2.4, 1.0 Hz, 1H, ic H), 6.92 (dd, J: 8.6, 0.7 Hz, 1H, aromatic H), 3.40 (s, 2H, NHZ) 1.48 (s, 9H, C(CH3)3). 13C NMR (101 MHz, CDC13)8 156.81, 155.65, , 138.69, 134.40, 129.10, 126.99, 120.85, 117.23, 116.69, 112.54, 34.89, 31.41. 4-(4-(tert-butyl)phen0xy)flu0r0aniline, I3-I 16°01.
Following ure B, I3-nI (501 mg, 1.73 mmol, 1.00 eq) was dissolved in MeOH (13 mL) and added to a suspension of Pd (10%) on activated carbon powder (56 mg, 0.05 mmol Pd, 0.03 eq) in MeOH (2 mL). The flask was purged with H2 (5X) and the reaction mixture was stirred at room ature for 1.5 h. The crude t was purified by flash column chromatography (SiOz; DCM) to give the title compound as colourless solid (455 mg, 1.75 mmol, quant. yield). Rf: 0.76 (DCM/MeOH 1%). HRMS (ESI) calcd. for C16H19FNO+ [M+H]+ 260.1445, found: 260.1442.1H NMR (400 MHz,CDC13)8 7.37 — 7.29 (m, 2H, aromatic H), 6.94 (t, J: 8.8 Hz, 1H, ic H), 6.91 — 6.85 (m, 2H, aromatic H), 6.52 (dd, J = 12.0, 2.7 Hz, 1H, aromatic H), 6.42 (ddd, J: 8.6, 2.7, 1.3 Hz, 1H, aromatic H), 3.61 (s, 2H, NH2), 1.33 (s, 9H, C(CH3)3). 13C NMR (101 MHz,CDC13)8 156.58, 156.44, 154.12, 145.02, 144.42, 144.33, , 134.71, 126.41, 123.92, 123.90, 115.43, 110.97, 110.93, 103.93, 103.72, 34.25, 31.59.
W0 93885 69 4-(4-cyclohexylphen0xy)flu0r0aniline, I3-J 55060., Following procedure B, I3-nJ (550 mg, 1.74 mmol, 1.00 eq) was dissolved in MeOH (12 mL) and added to a suspension of Pd (10%) on activated carbon powder (46 mg, 0.04 mmol Pd, 0.03 eq) in MeOH (3 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 2 h. The crude product was purified by flash column chromatography (SiOz; trolether 1:1 to 2:1) to give the title compound as pale rose solid (489 mg, 1.71 mmol, 98% yield). Rf: 0.81 (DCM/MeOH 4%). HRMS (ESI) calcd. for C18H21FNO+ [M+H]+ 02, found: 286.1613. 1H NMR (400 MHz, CDC13)5 7.19 ? 7.10 (m, 2H, aromatic H), 6.93 (t, J: 8.8 Hz, 1H, ic H), 6.90 — 6.83 (m, 2H, aromatic H), 6.51 (dd, J: 12.1, 2.7 Hz, 1H, aromatic H), 6.41 (ddd, J: 8.6, 2.7, 1.2 Hz, 1H, aromatic H), 3.66 (s, 2H, NH2), 2.52 — 2.45 (m, 1H, cyclohexyl H), 1.96 — 1.72 (m, 5H, cyclohexyl H), 1.53 — 1.18 (m, 5H, exyl H). 13C NMR (101 MHz,CDC13)5 156.74, 156.55, 154.09, 144.39, 144.30, 142.04, 134.86, 134.74, 127.79, 123.89, 115.76, , 110.90, 103.90, 103.69, 43.80, 34.72, 26.99, 26.22. 3-flu0r0(4-(tert-pentprhen0xy)am°line, I3-K Following procedure B, I3-nK (497 mg, 1.64 mmol, 1.00 eq) was dissolved in MeOH (13 mL) and added to a suspension of Pd (10%) on activated carbon powder (28 mg, 0.03 mmol Pd, 0.02 eq) in MeOH (2 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 1.5 h. The crude product was purified by flash column chromatography (SiOz; ethyl acetate/petrolether 1:10 to 1:75) to give the title compound as orange oil (463 mg, 1.69 mmol, quant. yield). Rf: 0.28 (EtOAc/petrolether 1:5). HRMS (ESI) calcd. for C17H21FNO+ [M+H]+ 274.1602, found: 274.1599. 1H NMR (400 MHz, CDClg) 5 7.26 — 7.17 (m, 2H, aromatic H), 6.92 (t, J: 8.8 Hz, 1H, aromatic H), 6.89 — 6.80 (m, 2H, aromatic H), 6.51 (dd, J: 12.0, 2.7 Hz, 1H, aromatic H), 6.42 (ddd, J: 8.7, 2.7, 1.2 Hz, 1H, aromatic H), 3.65 (s, 2H, NH2), 1.61 (q, J: 7.4 Hz, 2H, Ar-C(CH3)2CH2CH3), 1.26 (s, 6H, Ar-C(CH3)2CH2CH3), 0.69 (t, J: 7.4 Hz, 3H, Ar-C(CH3)2CH2CH3). 13c NMR (101 MHz, CDClg) 8 156.61, 156.37, 154.15, , 144.25, 143.34, 134.98, 134.86, 123.95, 123.92, , 110.98, 110.95, 104.00, 103.78, 37.07, 28.68, 9.26. 6-(4-(2-methylpentan-Z—prhen0xy)pyridinamine, I3-L Following procedure B, I3-nL (50 mg, 0.17 mmol, 1.00 eq) was dissolved in MeOH (12 mL) and added to a suspension of Pd (10%) on activated carbon powder (31 mg, 0.03 mmol Pd, 0.17 eq) in MeOH (3 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 1.5 h. The crude product was purified by flash column chromatography (SiOz; DCM/MeOH 1%) to give the title compound as pale orange oil (39 mg, 0.14 mmol, 87% yield). Rf: 0.41 (DCM/MeOH 4%). HRMS (ESI) calcd. for C17H23N20+ [M+H]+ 271.1805, found: 271.1796.1H NMR (400 C13)8 7.73 (d, J: 2.9 Hz, 1H, aromatic H), 7.32 — 7.26 (m, 2H, ic H), 7.07 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.01 — 6.94 (m, 2H, ic H), 6.74 (d, J: 8.6 Hz, 1H, aromatic H), 3.35 (s, 2H, NH2), 1.61 — 1.51 (m, 2H, C(CH3)2CH2CH2CH3), 1.27 (s, 6H, C(CH3)2CH2CH2CH3), 1.16 — 1.01 (m, 2H, C(CH3)2CH2CH2CH3), 0.82 (t, J: 7.3 Hz, 3H, C(CH3)2CH2CH2CH3). 13C NMR (101 MHz, CDC13) 8 156.82, 153.32, 145.10, 138.67, 134.32, 128.12, 127.09, 126.99, , 119.13, 112.53, 47.31, 37.49, 29.17, 18.08, 14.90. 4-(4-((3r,5r, 7r)-adamantan-I-prhenoxy)aniline, I3-M $0011., ing procedure B, I3-nM (615 mg, 1.76 mmol, 1.00 eq) was dissolved in toluene (15 mL) and added to a suspension of Pd (10%) on activated carbon powder (126 mg, 0.12 mmol Pd, 0.07 eq) in MeOH (10 mL). The flask was purged with H2 (6X) and the reaction mixture was d at room temperature for 19 h. The crude product was purified by flash column chromatography (SiOz; DCM) to give the title compound as beige solid (538 mg, 1.68 mmol, 96% yield). Rf: 0.39 (DCM/MeOH 1%). HRMS (ESI) calcd. for C22H26NO+ [M+H]+ 320.2009, found: 320.2006. 1H NMR (400 MHz, CDClg) 8 7.32 — 7.25 (m, 2H, aromatic H), 6.93 — 6.86 (m, 4H, aromatic H), 6.72 — 6.65 (m, 2H, aromatic H), 3.56 (s, 2H, NH2), 2.14 — 2.06 (m, 3H, adamantyl H), 1.90 (d, J: 2.9 Hz, 5H, adamantyl H), 1.84 — 1.71 (m, 5H, adamantylH). 13c NMR (101 MHz,CDC13)8 156.59, 149.02, 145.32, 142.51, 125.98, 121.10, , 116.34, 43.45, 36.88, 35.78, 29.07. (3r,5r, 7r)-adamantan-I-yl)phenoxy)flu0r0aniline, I3-N W‘11...
Following procedure B, I3-nN (570 mg, 1.55 mmol, 1.00 eq) was ved in toluene (10 mL) and added to a suspension of Pd (10%) on activated carbon powder (143 mg, 0.13 mmol Pd, 0.09 eq) in MeOH (10 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 7 h. The crude product was purified by flash column chromatography (SiOz; DCM) to give the title nd as colourless solid (510 mg, 1.51 mmol, 97% yield). Rf: 0.67 (DCM/MeOH 1%). HRMS (ESI) calcd. for C22H25FNO+ [M+H]+ 338.1915, found: 338.1916.1H NMR (400 MHz,CDC13)8 7.32 — 7.23 (m, 2H, aromatic H), 6.92 (t, J: 8.8 Hz, 1H, aromatic H), 6.89 — 6.81 (m, 2H, aromatic H), 6.51 (dd, J: 12.0, 2.7 Hz, 1H, aromatic H), 6.41 (ddd, J: 8.7, 2.7, 1.2 Hz, 1H, aromatic H), 3.64 (s, 2H, NH2), 2.11 — 2.07 (m, 3H, adamantyl H), 1.89 (d, J: 2.9 Hz, 5H, adamantyl H), 1.83 — 1.70 (m, 5H, adamantylH). 13c NMR (101 MHz,CDC13)8 156.60, 156.47, , 145.37, 144.33, 144.24, 134.91, 134.79, 125.98, 123.97, 123.95, 115.47, 110.98, 110.95, 103.98, 103.76, 43.43, 36.86, 35.76, 29.06. 6-(4-is0pr0pylphenoxy)pyridinamine, I3-0 WO 93885 72 Following procedure B, I3-nO (202 mg, 0.78 mmol, 1.00 eq) was added to a suspension of Pd (10%) on activated carbon powder (76 mg, 0.07 mmol Pd, 0.09 eq) in MeOH (15 mL). The flask was purged with H2 (6X) and the reaction mixture was stirred at room temperature for 1 h. The crude product was purified by flash column chromatography (Si02; DCM/MeOH 1%) to give the title compound as beige solid (150 mg, 0.66 mmol, 84% yield). Rf: 0.42 (DCM/MeOH 4%). HRMS (ESI) calcd. for C14H17N20+ [M+H]+ 229.1335, found: 229.1326. 1H NMR (400 MHz, CDClg) 5 7.70 (d, J: 2.9 Hz, 1H, aromatic H), 7.24 — 7.14 (m, 2H, aromatic H), 7.05 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 7.01 — 6.93 (m, 2H, ic H), 6.73 (d, J: 8.6 Hz, 1H, aromatic H), 3.33 (s, 2H, NH2), 2.89 (hept, J: 6.9 Hz, 1H, CH(CH3)2), 1.24 (d, .1: 7.0 Hz, 6H, CH(CH3)2). 13C NMR (101 MHz, CDC13)8 156.88, 153.59, , 138.68, 134.19, 127.55, 126.97, 119.76, 112.35, 77.48, 77.16, 76.84, 33.57, 24.20. 2,4,4—trimethylpentan-Z-yl)phenoxy)pyridinamine, I3-P Following procedure B, I3-nP (283 mg, 0.86 mmol, 1.00 eq) was dissolved in toluene (4 mL) and added to a suspension of Pd (10%) on activated carbon powder (78 mg, 0.07 mmol Pd, 0.09 eq) in MeOH (15 mL). The flask was purged with H2 (5X) and the reaction mixture was stirred at room temperature for 2 h. The crude t was d by flash column chromatography (SiOz; DCM/MeOH 2%) to give the title compound as colourless solid (235 mg, 0.79 mmol, 91% yield). Rf: 0.49 (DCM/MeOH 4%). HRMS (ESI) calcd. for C19H27N20+ [M+H]+ 299.2118, found: 299.2112.1H NMR (400 MHz,CDC13)5 7.73 (dd, J = 3.0, 0.7 Hz, 1H, ic H), 7.37 — 7.29 (m, 2H, aromatic H), 7.06 (dd, J: 8.6, 3.0 Hz, 1H, aromatic H), 6.99 — 6.93 (m, 2H, aromatic H), 6.71 (dd, J: 8.6, 0.7 Hz, 1H, aromatic H), 3.10 (s, 2H, NH2), 1.72 (s, 2H, Ar-C(CH3)2CH2C(CH3)3), 1.36 (s, 6H, Ar- C(CH3)2CH2C(CH3)3), 0.73 (s, 9H, Ar-C(CH3)2CH2C(CH3)3). 13c NMR (101 MHz, CDClg) 5 156.87, 153.39, 145.48, 138.70, 134.39, 127.37, 126.94, 118.96, 112.41, 57.20, 38.36, 32.50, 31.93, 31.68.

Claims (17)

1. The use of a compound having Notch signaling pathway inhibition properties, n the compound is selected from the group consisting of: Formula I O N 6-(4-Tert-Butylphenoxy)PyridinAmine, Formula II e tert-butyl)phenoxy)pyridinamine, Formula II f 4-(4-(tert-butyl)phenoxy)fluoroaniline, Formula II g 6-(4-(tert-Pentyl)phenoxy)pyridinamine, Formula II i 3-Fluoro(4-(tert-pentyl)phenoxy)aniline, Formula II where m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl-, Bror R1, R2, R3 and R4 are each independently selected from the group ting of H, isopropyl, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, Formula III where m is an integer selected from 1 to 4; n is an integer selected from 1 to 15; W is selected from H and halogens; the halogen is selected from F-, Cl-, Bror R4 and R15 are each independently selected from the group consisting of H, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, and Formula IV where m is an r selected from 1 to 4; W is selected from H and halogens; the halogen is ed from F-, Cl-, Bror R4 is independently ed from the group consisting of H, isopropyl, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, or one of their derivatives, salts, solvates, tautomers or isomers thereof, for the manufacture of a medicament for the treatment and/or prevention of a Notch dependent cancer.
2. The use according to claim 1 wherein said compound having Notch signaling pathway inhibition properties is selected from the group consisting of: 6-(4-Tert-Butylphenoxy)PyridinAmine (13) of formula I 4-(4-cyclohexylphenoxy)aniline of formula IIIa, Formula III a 6-(4-((3r,5r,7r)-adamantanyl)phenoxy)pyridinamine of formula IVa, Formula IV a 6-(3-(tert-butyl)phenoxy)pyridinamine of formula IIe, Formula II e tert-butyl)phenoxy)fluoroaniline of formula IIf, Formula II f 6-(4-(tert-Pentyl)phenoxy)pyridinamine of formula IIg, Formula II g 6-(4-Butylphenoxy)pyridinamine of formula IIh, Formula II h 3-Fluoro(4-(tert-pentyl)phenoxy)aniline of a II i, Formula II i 4-(4-Cyclohexylphenoxy)fluoroaniline of a III b Formula III b 4-(4-((3r,5r,7r)-Adamantanyl)phenoxy)aniline of formula IVb, Formula IV b 6-(4-cyclohexylphenoxy)pyridinamine of formula IIIc, a III c or one of its salts, solvates, tautomers, or stereoisomers thereof.
3. The use of claim 1 or 2 wherein the Notch dependent cancer is selected from the group sing T cell-Acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), chronic lymphocytic ia (CLL), Mantle cell lymphoma (MCL), breast cancer, pancreatic cancer, prostate cancer, melanoma, brain tumors, tumor angiogenesis, and colorectal cancer.
4. The use of any one of claims 1-3 wherein the Notch dependent cancer is resistant to γ-secretase inhibitor treatment.
5. A pharmaceutical composition comprising a compound having Notch signaling pathway inhibition properties, n the compound is selected from the group consisting of: 6-(4-((3r,5r,7r)-adamantanyl)phenoxy)pyridinamine of formula IVa, Formula IV a 6-(4-Butylphenoxy)pyridinamine of formula IIh, Formula II h 4-(4-Cyclohexylphenoxy)fluoroaniline of formula III b Formula III b 6-(4-cyclohexylphenoxy)pyridinamine of formula IIIc, Formula III c or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers thereof, and a pharmaceutically acceptable carrier.
6. A kit comprising one or more doses of a compound having Notch signaling pathway tion ties, wherein the nd is selected from the group consisting of: 6-(4-((3r,5r,7r)-adamantanyl)phenoxy)pyridinamine of formula IVa, Formula IV a 6-(4-Butylphenoxy)pyridinamine of formula IIh, Formula II h 4-(4-Cyclohexylphenoxy)fluoroaniline of formula III b, Formula III b 6-(4-cyclohexylphenoxy)pyridinamine of formula IIIc, a III c or ceutically acceptable salts, solvates, tautomers or stereoisomers thereof; optionally with reagents and/or instructions for use.
7. The kit of claim 6, further comprising one or more doses of a chemotherapeutic agent.
8. The use of any one of claims 1-4, for inhibiting in vitro the Notch signalling pathway in cells.
9. The use of claim 8 wherein the cells are cancer cells.
10. The use of any one of claims 1-4, wherein the Notch dependent cancer is treated and/or ted by inhibiting in vivo the Notch signalling pathway in cells.
11. The use of claim 10 wherein the cells are cancer cells.
12. The use of any one of claims 1 to 4, wherein said treatment of a Notch dependent cancer ses the steps of: i) determining in cancer cells obtained from a biological sample of a subject whether the cancer is Notch signalling pathway dependent, and ii) treating said subject based upon whether the cancer is Notch dependent cancer by administering a therapeutically effective amount of the compound of any one of claims 1-4, or a pharmaceutical composition of claim 5.
13. The use of claim 12 wherein the Notch ling pathway dependency in cancer cells is determined by an in vitro γ-secretase complex activity assay.
14. The use of claim 12 or 13 wherein said treatment of a Notch dependent cancer further comprises administering at least one conventional cancer treatment.
15. The use of claim 14 n the conventional cancer treatment is administered , simultaneously or after the administration of the therapeutically effective amount of the compound of any one of claims 1-4, or the pharmaceutical ition of claim 5.
16. The use of claim 14 or 15 wherein the conventional cancer treatment consists of radiotherapy and/or chemotherapy.
17. The use of the a compound selected from the group consisting of Formula I O N 6-(4-Tert-Butylphenoxy)PyridinAmine, Formula II e 6-(3-(tert-butyl)phenoxy)pyridinamine, Formula II f 4-(4-(tert-butyl)phenoxy)fluoroaniline, a II g 6-(4-(tert-Pentyl)phenoxy)pyridinamine, Formula II h 6-(4-Butylphenoxy)pyridinamine, Formula II i 3-Fluoro(4-(tert-pentyl)phenoxy)aniline, Formula II where m is an integer selected from 1 to 4; W is selected from H and halogens; the halogen is selected from F-, Cl-, Bror R1, R2, R3 and R4 are each independently ed from the group consisting of H, isopropyl, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, Formula III where m is an integer selected from 1 to 4; n is an integer selected from 1 to 15; W is selected from H and halogens; the halogen is selected from F-, Cl-, Bror R4 and R15 are each ndently selected from the group consisting of H, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, and Formula IV where m is an r selected from 1 to 4; W is selected from H and halogens; the n is selected from F-, Cl-, Bror R4 is independently selected from the group consisting of H, isopropyl, tertbutyl, (CH2)nCH3; the subscript n is an integer independently selected from 1 to 15; X is O, NR7 where is R7 is H; Y is N or CH; Z is NR10R11 where R10 and R11 is H, or one of its salts, solvates, tautomers, or stereoisomers thereof, for the manufacture of a medicament for the treatment of a disease associated with an up-regulated Notch signalling pathway activity, wherein the treatment comprises stering the compound to a subject in need thereof. A} [M ; Ni cggufiure a} MOD iiansfecied HeLa ceiis luezifarasm 3 Aucif ve Eatwe (3 q“. L‘I-E 1% E ‘32 (I n 25K} DAPT (um; .4. 'M _. m azciiviiy Q aciav'iy ~‘ Euciferase 53 :3: feaaw c: ' o: S3 a: Lit 5:: (n Reia‘u‘va 0 4:. Reliwe C) 4?' :9w Q N: C: 1:) W‘WM’WWK
NZ627559A 2011-12-21 2012-12-21 Inhibitors of notch signalling pathway and use thereof in treatment of cancers NZ627559B2 (en)

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