Patent Application
20210309615
The present invention relates to phenyl amino pyrimidine compounds which are inhibitors of protein kinases, including JAK kinases. In particular the compounds are selective for JAK2 kinases. The kinase inhibitors can be used in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
JAKs are kinases which phosphorylate a group of proteins called Signal Transduction and Activators of Transcription or STATs. When phosphorylated, STATs dimerize, translocate to the nucleus and activate expression of genes which lead to, amongst other things, cellular proliferation.
The central role played by the JAK family of protein tyrosine kinases in the cytokine dependent regulation of both proliferation and end function of several important cell types indicates that agents capable of inhibiting the JAK kinases are useful in the prevention and chemotherapeutic treatment of disease states dependent on these enzymes. Potent and specific inhibitors of each of the currently known four JAK family members will provide a means of inhibiting the action of the cytokines that drive immunological and inflammatory diseases.
Myeloproliferative disorders (MPD) include, among others, polycythemia vera (PV), primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis (IMF), chronic myelogenous leukemia (CML), systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodisplastic syndrome (MDS) and systemic mast cell disease (SMCD). JAK2 is a member of the JAK family of kinases in which a specific mutation (JAK2V617F) has been found in 99% of polycythemia vera (PV) patients and 50% of essential thrombocytopenia (ET) and idiopathic myelofibrosis (MF). This mutation is thought to activate JAK2, giving weight to the proposition that a JAK2 inhibitor will be useful in treating these types of diseases.
Asthma is a complex disorder characterized by local and systemic allergic inflammation and reversible airway obstruction. Asthma symptoms, especially shortness of breath, are a consequence to airway obstruction, and death is almost invariably due to asphyxiation. Airway Hyper Responsiveness (AHR), and mucus hyper secretion by goblet cells are two of the principle causes of airway obstruction in asthma patients. Intriguingly recent work in animal experimental models of asthma has underscored the importance of IL-13 as a key player in the pathology of asthma. Using a specific IL-13 blocker, it has been demonstrated that IL-13 acts independently of IL-4 and may be capable of inducing the entire allergic asthma phenotype, without the induction of IgE (i.e., in a non-atopic fashion). This and other models have pointed to an important second tier mechanism for elicitating the pathophysiology of asthma, that is not dependent on the production of IgE by resident B-cells or the presence of eonisophils. A direct induction of AHR by IL-13, represents an important process that is likely to be an excellent target for intervention by new therapies. A contemplated effect of a JAK2 inhibitor to the lungs would result in the suppression of the local release of IL-13 mediated IgE production, and therefore reduction in histaminine release by mast cells and eosinophils. This and other consequences of the absence of IL-13 indicate that many of the effects of asthma may be alleviated through administration of a JAK2 inhibitor to the lungs.
Chronic Obstructive Pulmonary Disease (COPD) is a term which refers to a large group of lung diseases which can interfere with normal breathing. Current clinical guidelines define COPD as a disease state characterized by airflow limitation which is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, particularly cigarette smoke and pollution. Several studies have pointed to an association between increased production of IL-13 and COPD, lending support to the proposition that the potential alleviation of asthma symptoms by use of a JAK2 inhibitor, may also be achieved in COPD. COPD patients have a variety of symptoms including cough, shortness of breath, and excessive production of sputum. COPD includes several clinical respiratory syndromes including chronic bronchitis and emphysema.
Chronic bronchitis is a long standing inflammation of the bronchi which causes increased production of mucus and other changes. The patient's symptoms are cough and expectoration of sputum. Chronic bronchitis can lead to more frequent and severe respiratory infections, narrowing and plugging of the bronchi, difficult breathing and disability.
Emphysema is a chronic lung disease which affects the alveoli and/or the ends of the smallest bronchi. The lung loses its elasticity and therefore these areas of the lungs become enlarged. These enlarged areas trap stale air and do not effectively exchange it with fresh air. This results in difficult breathing and may result in insufficient oxygen being delivered to the blood. The predominant symptom in patients with emphysema is shortness of breath.
Additionally, there is evidence of STAT activation in malignant tumors, among them lung, breast, colon, ovarian, prostate and liver cancer, as well as Hodgkin's lymphoma, multiple myeloma and hepatocellular carcinoma. Chromosomal translocations involving JAK2 fusions to Tel, Bcr and PCM1 have been described in a number of hematopoietic malignancies including chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic eosinophilic leukemia (CEL), myelodisplastic syndrome (MDS), myeloproliferative disease (MPD) and acute lymphocytic leukemia (ALL). This suggests treatment of hyperproliferative disorders such as cancers including multiple myeloma; prostate, breast and lung cancer; Hodgkin's Lymphoma; CML; AML; CEL; MDS; ALL; B-cell Chronic Lymphocytic Leukemia; metastatic melanoma; glioma; and hepatoma, by JAK inhibitors is indicated.
Potent inhibitors of JAK2, in addition to the above, will also be useful in vascular disease such as hypertension, hypertrophy, cardiac ischemia, heart failure (including systolic heart failure and diastolic heart failure), migraine and related cerebrovascular disorders, stroke, Raynaud's phenomenon, POEMS syndrome, Prinzmetal's angina, vasculitides, such as Takayasu's arteritis and Wegener's granulomatosis, peripheral arterial disease, heart disease and pulmonary arterial hypertension.
Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease affecting the pulmonary arterioles resulting in an elevation in pulmonary artery pressure and pulmonary vascular resistance but with normal or only mildly elevated left-sided filling pressures. PAH is caused by a constellation of diseases that affect the pulmonary vasculature. PAH can be caused by or associated with collagen vascular disorders such as systemic sclerosis (scleroderma), uncorrected congenital heart disease, liver disease, portal hypertension, HIV infection, Hepatitis C, certain toxins, splenectomy, hereditary hemorrhagic teleangiectasia, and primary genetic abnormalities. In particular, a mutation in the bone morphogenetic protein type 2 receptor (a TGF-b receptor) has been identified as a cause of familial primary pulmonary hypertension (PPH). It is estimated that 6% of cases of PPH are familial, and that the rest are “sporadic.” The incidence of PPH is estimated to be approximately 1 case per 1 million population. Secondary causes of PAH have a much higher incidence. The pathologic signature of PAH is the plexiform lesion of the lung which consists of obliterative endothelial cell proliferation and vascular smooth muscle cell hypertrophy in small precapillary pulmonary arterioles. PAH is a progressive disease associated with a high mortality. Patients with PAH may develop right ventricular (RV) failure. The extent of RV failure predicts outcome. The JAK/STAT pathway has recently been implicated in the pathophysiology of PAH. JAKs are kinases which phosphorylate a group of proteins called Signal Transduction and Activators of Transcription or STATs. When phosphorylated, STATs dimerize, translocate to the nucleus and activate expression of genes which lead to proliferation of endothelial cells and smooth muscle cells, and cause hypertrophy of cardiac myocytes. There are three different isoforms of JAK: JAK1, JAK2, and JAK3. Another protein with high homology to JAKs is designated TYK2. An emerging body of data has shown that the phosphorylation of STAT3, a substrate for JAK2, is increased in animal models of PAH. In the rat monocrotaline model, there was increased phosphorylation of the promitogenic transcription factor STAT3. In this same study pulmonary arterial endothelial cells (PAECs) treated with monocrotaline developed hyperactivation of STAT3. A promitogenic agent or protein is an agent or protein that induces or contributes to the induction of cellular proliferation. Therefore, one effect of JAK2 inhibition would be to decrease proliferation of endothelial cells or other cells, such as smooth muscle cells. A contemplated effect of a JAK2 inhibitor would be to decrease the proliferation of endothelial cells or other cells which obstruct the pulmonary arteriolar lumen. By decreasing the obstructive proliferation of cells, a JAK2 inhibitor could be an effective treatment of PAH.
Additionally the use of JAK kinase inhibitors for the treatment of viral diseases and metabolic diseases is indicated.
Although the other members of the JAK family are expressed by essentially all tissues, JAK3 expression appears to be limited to hematopoetic cells. This is consistent with its essential role in signalling through the receptors for IL-2, IL4, IL-7, IL-9 and IL-15 by non-covalent association of JAK3 with the gamma chain common to these multichain receptors. Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. An XSCID syndrome in which patients with either mutated or severely reduced levels of JAK3 protein has been identified, suggesting that immunosuppression should result from blocking signalling through the JAK3 pathway. Gene Knock out studies in mice have suggested that JAK3 not only plays a critical role in B and T lymphocyte maturation, but that JAK3 is constitutively required to maintain T cell function. Taken together with the biochemical evidence for the involvement of JAK3 in signalling events downstream of the IL-2 and IL-4 receptor, these human and mouse mutation studies suggest that modulation of immune activity through the inhibition of JAK3 could prove useful in the treatment of T-cell and B-cell proliferative disorders such as transplant rejection and autoimmune diseases. Conversely undesired inhibition of JAK3 could have a devastating effect on the immune status of an individual treated with drug.
Although the inhibition of various types of protein kinases, targeting a range of disease states, is clearly beneficial, it has been to date demonstrated that the identification of a compound which is selective for a protein kinase of interest, and has good “drug like” properties such as high oral bioavailability, is a challenging goal. In addition, it is well established that the predictability of inhibition, or selectivity, in the development of kinase inhibitors is quite low, regardless of the level sequence similarity between the enzymes being targeted.
The challenges in developing therapeutically appropriate JAK2 inhibitors for use in treatment kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases include designing a compound with appropriate specificity which also has good drug-likeliness.
There is therefore a continuing need to design and/or identify compounds which specifically inhibit the JAK family of kinases, and particularly compounds which may preferentially inhibit one of the JAK kinases relative to the other JAK kinases, particularly JAK2. There is a need for such compounds for the treatment of a range of diseases.
In a first aspect, there is provided a compound of formula I
wherein
Q and Z are independently selected from N and CR1;
n is 1, 2 or 3;
R1 is independently selected from hydrogen, halogen, R2, OR2, OH, R4, OR4, CN, CF3, (CH2)˜N(R2)2, NO2, R2R4, SO2R4, NR2SO2R3, COR4, NR2COR3, CO2H, CO2R2, NR2COR4, R2CN, R2CN, R2OH, R2OR3 and OR5R4; or
two R1 substituents together with the carbons which they are attached to form an unsaturated 5 or 6 membered heterocyclyl;
R2 is substituted or unsubstituted C1-4alkyl or substituted or unsubstituted C1-4 alkylene where up to 2 carbon atoms can be optionally replaced with CO, NRY, CONRY, S, SO2 or O;
R3 is R2, C2-4alkenyl or substituted or unsubstituted aryl;
R4 is NH2, NHR2, N(R1)2, substituted or unsubstituted morpholino, substituted or unsubstituted thiomorpholino, substituted or unsubstituted thiomorpholino-1-oxide, substituted or unsubstituted thiomorpholino-1, 1-dioxide, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperidinyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted tetrahydrofuranyl and substituted or unsubstituted tetrahydropyranyl;
R5 is substituted or unsubstituted C1-4alkylene;
R6-R10 are independently selected from H, RXCN, halogen, substituted or unsubstituted C1-4alkyl, OR1, CO2R1, N(R1)2, NO2, CON(R1)2, SO2N(RY)2, N(SO2R1)2, substituted or unsubstituted piperazinyl, N(RY)SO2R2 and CF3;
Rx is absent or substituted or unsubstituted C1-6alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R1, NRY, CONRY, S, SO2 or O;
RY is H or substituted or unsubstituted C1-4alkyl; and
R11 is selected from H, halogen, substituted or unsubstituted C1-4 alkyl, OR2, CO2R2, CN, CON(R1)2 and CF3,
or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.
In a second aspect, there is provided a process for the preparation of the compound of formula I defined above which comprises the step of coupling a compound of formula II
wherein
Y and R11 and n are as defined above and X is a leaving group with compounds of formulae III and IV
wherein
n, Z, R1 and R6-R10 are as defined above; and
M is B or a metal such as Sn, Zn or Mg.
The compounds of formula I are kinase inhibitors, preferably JAK inhibitors, more preferably JAK2 inhibitors. These compounds are useful in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
In a third aspect, there is provided a pharmaceutical agent or metabolites thereof comprising the compound of formula I defined above.
There is also provided use of the compound of formula I as a pharmaceutical agent or metabolites thereof.
There is further provided the compound of formula I defined above for use as a pharmaceutical agent or metabolites thereof.
In a fourth aspect, there is provided a kinase inhibitor comprising the compound formula I defined above.
There is also provided use of the compound of formula I defined above as a kinase inhibitor.
There is further provided the compound of formula I defined above for use as a kinase inhibitor.
In a fifth aspect, there is provided a compound of formula 1 defined above for use as a pharmaceutical agent or metabolites thereof, preferably a kinase inhibitor, more preferably a JAK kinase inhibitor, most preferably a JAK2 selective inhibitor.
The compound of formula I may also be administered in the form of a pharmaceutical composition together with a pharmaceutically acceptable carrier.
In a sixth aspect, there is provided a pharmaceutical composition comprising the compound of formula I defined above and a pharmaceutically acceptable carrier.
In one embodiment, the pharmaceutical composition also comprises one or more additional therapeutic agents.
The compound of formula I may be contained within or attached to an implant, such as a drug eluting stent. For example, when the compound is used for the treatment of pulmonary arterial hypertension (PAH), the compound may be contained within or attached to a pulmonary artery stent, which may act locally, or be released from the stent into the pulmonary circulation where the compound exerts its therapeutic activity in the pulmonary vasculature.
In a seventh aspect, there is provided an implant which comprises the compound of formula I defined above.
In an eighth aspect, there is provided a method for the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases which comprises administering an effective amount of the compound of formula I or a pharmaceutical composition defined above to a subject in need thereof.
There is also provided use of the compound of formula I or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
There is further provided use of the compound of formula I or a pharmaceutical composition as defined above in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
There is still further provided the compound of the formula I or a pharmaceutical composition defined above for use in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
In a ninth aspect, there is provided a method of inhibiting a kinase in a cell comprising contacting the cell with the compound of formula I defined above.
The present invention relates to compounds of formula I which inhibit kinases, in particular JAK kinases such as JAK2 and are useful in the treatment of kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
Compounds
The present invention relates to compounds of formula I
wherein
Q and Z are independently selected from N and CR1;
n is 1, 2 or 3;
R1 is independently selected from hydrogen, halogen, R2, OR2, OH, R4, OR4, CN, CF3, (CH2)˜N(R2)2, NO2, R2R4, SO2R4, NR2SO2R3, COR4, NR2COR3, CO2H, CO2R2, NR2COR4, R2CN, R2CN, R2OH, R2OR3 and OR5R4; or
two R1 substituents together with the carbons which they are attached to form an unsaturated 5 or 6 membered heterocyclyl;
R2 is substituted or unsubstituted C1-4alkyl or substituted or unsubstituted C1-4 alkylene where up to 2 carbon atoms can be optionally replaced with CO, NRY, CONRY, S, SO2 or O;
R3 is R2, C2-4alkenyl or substituted or unsubstituted aryl;
R4 is NH2, NHR2, N(R1)2, substituted or unsubstituted morpholino, substituted or unsubstituted thiomorpholino, substituted or unsubstituted thiomorpholino-1-oxide, substituted or unsubstituted thiomorpholino-1, 1-dioxide, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperidinyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted tetrahydrofuranyl and substituted or unsubstituted tetrahydropyranyl;
R5 is substituted or unsubstituted C1-4alkylene;
R6-R10 are independently selected from H, RXCN, halogen, substituted or unsubstituted C1-4alkyl, OR1, CO2R1, N(R1)2, NO2, CON(R1)2, SO2N(RY)2, N(SO2R1)2, substituted or unsubstituted piperazinyl, N(RY)SO2R2 and CF3;
Rx is absent or substituted or unsubstituted C1-6alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R1, NRY, CONRY, S, SO2 or O;
RY is H or substituted or unsubstituted C1-4alkyl; and
R11 is selected from H, halogen, substituted or unsubstituted C1-4 alkyl, OR2, CO2R2, CN, CON(R1)2 and CF3, or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.
In one embodiment, the compound of formula I has the formula Ia:
wherein,
Q and Z are independently selected from N and CR1;
R1 is independently selected from H, halogen, R2, OR2, OH, R4, CN, CF3, NO2, R2R4, SO2R4, NR2SO2R3, COR4, CO2H, CO2R2, NR2COR3, NR2COR4, R2CN, R2OH, R2OR3 and OR5R3; or
two R1 substitutents together with the carbon atoms to which they are attached form an unsaturated N-containing 5 or 6-membered heterocyclyl;
R2 is C1-4alkyl, or C1-4alkylene;
R3 is R2, C2-4alkenyl or aryl;
R4 is NH2, NHR2, N(R2)2, morpholino, thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1, 1-dioxide, 4-carbonylmethyl piperazinyl, 4-methyl piperazinyl, 3- or 4-hydroxy piperidinyl, 4 hydroxymethyl piperidinyl, 4-pyrrolidinyl piperidinyl, 4 or 5-methyl oxazolyl, 4-hydroxy pyridinyl, 3-hydroxy pyrrolyl, 3-hydroxy pyrrolidinyl, pyridinyl pyrazolyl or imidazolyl;
R5 is C2-4alkylene;
R6-R9 are independently selected from H, RXCN, halogen, substituted or unsubstituted C1-4alkyl, substituted or unsubstituted aryl, OR1, CO2R1, N(R1)2, NO2, CON(R1)2 and CON(R1)2;
RX is substituted or unsubstituted C1-4alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R1, NRY, CONRY, SO, SO2, or O;
RY is H or substituted or unsubstituted C1-4alkyl; and
R1 is selected from H, halogen, substituted or unsubstituted C1-4 alkyl, OR2, CO2R2, CN, CON(R1)2 and CF3, or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.
Preferably Q is N and Z is CR1.
Preferably R1 is hydrogen, morpholinyl, CH2morpholinyl, C1-4alkoxy, thiomorpholinyl, 3-hydroxypyrrolidinyl, iodo, fluoro, OH, 4-hydroxy piperidinyl, 4 hydroxymethyl piperidinyl, N-methyl piperidinyl, 3-hydroxy piperidinyl, carbonyl 4-pyrrolidinyl piperidinyl, oxy-4-piperidinyl, 4-carbonylmethyl piperazinyl, 4-methyl piperzinyl, 4-NHSO2CH3-piperidinyl, 4-oxy piperidinyl, imidazolyl CON(R1)2, CF3 or R2OR3.
Preferably R6 is H or methyl.
Preferably R7 is H, methyl, methoxy, halogen such as chloro or hydroxy.
Preferably R8 is H, RXCN such as CONHCN, CH2NHCOCN, CN, CONHC(CH3)2CN, NCNSO2CH3, SO2NHCH2CN or N(SO2CH3)CH2CN, OH, CO2CH2CH3, CON(R1)2, N(R1)2 or CO2R1.
Preferably R9 is H, RXCN such as CONHCN, CH2NHCOCN or CH2NHCN, methoxy halogen, OCF3 or CF3.
Preferably R1 is H, halogen, substituted or unsubstituted C1-4alkyl, OR2, CO2R2, CN or CF3, more preferably H, methyl, methoxy, Cl, Br, F or CO2R2, most preferably H or methyl.
In a preferred embodiment, the compound of formula I or Ia has the formula Ib:
wherein
Z is independently selected from N and CH;
R1 is independently selected from H, halogen, OH, CONHR2, CON(R2)2, CF3, R2OR2, CN, morpholino, thiomorpholinyl, thiomorpholino-1, 1-dioxide, substituted or unsubstituted piperidinyl, substituted or unsubstituted piperazinyl, imidazolyl, substituted or unsubstituted pyrrolidinyl and C1-4alkylene wherein the carbon atoms are optionally replaced with NRY and/or O substituted with morpholino, thiomorpholinyl, thiomorpholino-1, 1-dioxide, substituted or unsubstituted piperidinyl, substituted or unsubstituted piperazinyl, imidazolyl or substituted or unsubstituted pyrrolidinyl;
R2 is substituted or unsubstituted C1-4alkyl;
RY is H or substituted or unsubstituted C1-4alkyl;
R8 is RXCN;
RX is substituted or unsubstituted C1-4alkylene wherein up to 2 carbon atoms can be optionally replaced with CO, NSO2R1, NRY, CONRY, SO, SO2 or O;
R11 is H or C1-4alkyl,
or an enantiomer thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.
Examples of compounds of formula I include, but are not limited to, the following:
1H NMR
1H NMR (300 MHz, d6- DMSO): δ 9.49 (1H, s), 8.54 (1H, d, 5.0 Hz), 8.27 (2H, d, J = 8.7 Hz), 8.10 (2H, d, J = 8.7 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.38 (1H, d, J = 5.0 Hz), 6.93 (2H, d, J = 8.7 Hz), 4.35 (2H, q, J = 6.9 Hz), 3.73 (4H, m), 3.04 (4H, m), 1.34 (3H, t, J = 6.9 Hz).
1H NMR (300 MHz, d6- DMSO): δ 9.46 (1H, s), 9.34 (1H, s), 8.60 (1H, s), 8.53 (1H, d, J = 5.1 Hz), 8.32 (1H, d, J = 7.8 Hz), 7.99 (1H, d, J = 7.8 Hz), 7.67 (3H, m), 7.68 (1H, d, J = 5.1 Hz), 6.92 (2H, d, J = 9.0 Hz), 4.37 (2H, brs), 3.74 (4H, m), 3.04 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.47 (1H, s), 9.32 5.5 Hz), 8.54 (1H, d, J = 5.0 Hz), 8.27 (2H, d, J = 8.7 Hz), 8.02 (2H, d, J = 8.2 Hz), 7.67 (2H, d, J = 9.1 Hz), 7.41 (1H, d, J = 5.5 Hz), 6.93 (2H, d, J = 9.1 Hz), 4.36 (2H, d, J = 5.5 Hz), 3.75 (4H, m), 3.05 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 8.42 (1H, d, J = 5.2 Hz), 8.21 (2H, d, J = 8.4 Hz), 7.95 (2H, d, J = 8.4 Hz), 7.60 (2H, d, J = 9.0 Hz), 7.27 (1H, d, J = 5.2 Hz), 7.27 (1H, d, J = 5.2 Hz), 6.98 (2H, d, J = 9.0 Hz), 3.84 (4H, m), 3.73 (2H, t, J = 5.8 Hz), 3.53 (2H, t, J = 5.8 Hz), 3.11 (4H, m).
1H NMR (300 MHz, CDCl3): δ 8.49 (d, J = 5.1 Hz, 1H), 8.37- 8.36 (m, 1H), 8.28-8.25 (m, 1H), 7.78-7.75 (m, 1H), 7.63- 7.61 (m, 1H), 7.57-7.54 (m, 2H), 7.09 (d, J = 4.8 Hz, 1H), 7.00-6.97 (m, 2H), 3.89 (t, J = 4.5 Hz, 4H), 3.16 (t, J = 4.9 Hz, 4H).
1H NMR (300 MHz, CDCl3): δ 8.50 (d, J = 5.1 Hz, 1H), 8.15 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.58-7.55 (m, 2H), 7.13-7.11 (m, 1H), 7.01-6.98 (m, 2H), 3.90 (t, J = 4.5 Hz, 4H), 3.16 (t, J = 4.2 Hz, 4H).
1H NMR (300 MHz, CDCl3): δ 8.49 (d, J = 5.7 Hz, 1H), 8.36 (dd, J = 5.7, 2.4 Hz, 1H), 8.29 (m, 1H), 7.54 (d, J = 9.3 Hz, 1H), 7.33 (t, J = 9.0 Hz, 1H), 7.10 (br. s, 1H), 7.05 (d, J = 5.1 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 3.88 (t, J = 5.1 Hz, 4H), 3.15 (t, J = 5.4 Hz, 4H).
1H NMR (300 MHz, CDCl3): δ 8.53 (d, J = 5.4 Hz, 1H), 8.44 (dd, J = 5.7 Hz, 2.1 Hz, 1H), 8.29 (m, 1H), 7.34 (t, J = 8.7 Hz, 1H), 7.19 (br. s, 1H), 7.12 (d, J = 5.1 Hz, 1H), 7.00 (s, 2H), 3.92 (s, 6H), 3.85 (s, 3H).
1H NMR (300 MHz, CDCl3): δ 8.39 (m, 1H), 8.23 (d, J = 1.8 Hz, 1H), 8.11 (dd, J = 8.7, 2.1 Hz, 1H), 7.57-7.55 (m, 2H), 7.05-7.02 (m, 2H), 7.01-6.90 (m, 2H), 3.89 (t, J = 4.5 Hz, (m, 1H), 3.15-3.13 (m, 4H).
1H NMR (300 MHz, CDCl3): δ 8.45 (1H, d, J = 5.0 Hz), 7.72 (1H, d, J = 1.6 Hz), 7.76 (1H, dd, J = 1.6, 8.0 Hz), 7.52 (3H, m), 7.14 (1H, s), 6.91 (2H, d, J = 9.0 Hz), 6.77 (1H, d, J = 5.0 Hz), 6.67 (1H, t, J = 5.7 Hz), 4.39 (2H, d, J = 5.7 Hz), 3.86 (4H, m), 3.11 (4H, m), 2.48 (3H, s).
1H NMR (300 MHz, CDCl3: d4-MeOH): δ 8.42 (1H, d, J = 5.2 Hz), 7.99 (1H, brs), 7.96 (1H, dd, J = 1.2, 8.1 Hz), 7.62 (2H, d, J = 9.2 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.19 (1H, d, J = 5.2 Hz), 6.99 (2H, d, J = 9.2 Hz), 4.33 (2H, s), 3.89 (4H, m), 3.15 (4H, m), 2.54 (3H, s). * Partially obscured by CHCl3 signal.
1H NMR (300 MHz, d6- DMSO): δ 9.40 (1H, s), 8.78 (1H, dd, J = 5.5, 5.9 Hz), 8.48 (1H, d, J = 5.5 Hz), 8.03 (2H, m), 7.67 (2H, d, J = 9.1 Hz), 7.50 (1H, t, J = 7.8 Hz), 7.43 (1H, m), 7.29 (1H, d, J = 5.0 Hz), 6.93 (2H, d, J = 9.1 Hz), 4.39 (2H, d, J = 5.9 Hz), 3.73 (4H, m), 3.71 (2H, s), 3.04 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.41 (1H, s), 8.47 (1H, d, J = 5.0 Hz), 8.13 (1H, brs), 8.02 (1H, ddd, J = 1.8, 4.1, 5.0 Hz), 7.68 (2H, d, J = 9.1 Hz), 7.49 (2H, brd, J = 4.5 Hz), 7.31 (1H, d, J = 5.0 Hz), 6.92 (2H, d, J = 9.1 Hz), 3.85 (2H, d, J = 5.9 Hz), 3.73 (4H, m), 3.63 (2H, d, J = 7.3 Hz), 3.03 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 10.46 (1H, s), 9.41 (1H, s), 8.53 (1H, s), 8.49 (1H, d, J = 5.5 Hz), 7.83 (1H, d, J = 7.8 Hz), 7.72 (2H, d, J = 9.1 Hz), 7.58 (1H, brd, J = 8.2 Hz). 7.48 (1H, dd, J = 7.8, 7.8 Hz), 7.24 (1H, d, J = 5.0 Hz), 6.96 (2H, d, J = 9.1 Hz), 3.95 (2H, 3.73 (4H, m), 3.04 (4H, m).
1H NMR (300 MHz, d6- acetonitrile): δ 8.42 (1H, d, J = 5.0 Hz), 7.72 (1H, br), 7.64 (2H, d, J = 9.1 Hz), 7.51-7.54 (2H, m), 7.37 (1H, dd, J = 7.8, 8.2 Hz), 7.20 (1H, d, J = 5.0 Hz), 6.98 (2H, m), 6.90 (1H, m), 5.04 (1H, t, J = 6.9 Hz), 4.22 (2H, d, J = 6.9 Hz), 3.79 (4H, m), 3.08 (4H, m).
1H NMR (300 MHz, CDCl3: δ 8.50 (d, J = 5.1 Hz, 1H), 7.79 (d, J = 1.2 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.61 (dd, J = 1.4, 8.0 Hz, 1H), 7.56 (d, J = 9.0 Hz, 2H), 7.12 (br. s, 1H), 7.10 (d, J = 5.4 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 4.05 (s, 3H), 3.89 (m, 4H), 3.14 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.47 (1H, s), 9.24 (1H, t, J = 5.9 Hz), 8.52 (1H, d, J = 5.5 Hz), 8.51 (2H, m), 8.24 (2H, d, J = 8.2 Hz), 8.05 (2H, d, J = 8.7 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.39 (1H, d, J = 5.5 Hz), 7.32 (2H, d, J = 5.9 Hz), 6.92 (2H, d, J = 9.1 Hz), 4.52 (2H, d, J = 5.9 Hz), 3.74 (4H, m), 3.04 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.45 (1H, s), 9.20 (1H, t, J = 5.9 Hz), 8.57 (1H, d, J = 1.8 Hz), 8.52 (1H, d, J = 5.0 Hz), 8.46 (1H, dd, J = 1.8, 5.0 Hz), 8.23 (2H, d, J = 8.7 Hz), 8.03 (2H, d, J = 8.7 Hz), 7.74 (1H, ddd, J = 1.8, 2.8, 7.8 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.38 (1H, d, J = 5.0 Hz), 7.36 (1H, m), 6.92 (2H, d, J = 9.1 Hz), 4.52 (2H, d, J = 5.9 Hz), 3.73 (4H, m), 3.04 (4H, m).
1H NMR (300 MHz, CDCl3): δ 8.51 (d, J = 5.1 Hz, 1H), 8.21 (s, 1H), 8.02 (d, J = 9.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.16 (br. s, 1H), 7.09 (d, J = 5.1 Hz, 1H), 6.96 (d, J = 9.0 Hz, 2H), 3.88 (m, 4H), 3.15 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.59 (1H, s), 9.32 (1H, t, J = 5.5 Hz), 8.59 (1H, d, J = 5.0 Hz), 8.31 (2H, d, J = 8.7 Hz), 8.02 (2H, d, J = 8.7 Hz), 7.47 (1H, d, J = 5.0 Hz), 7.30 (2H, s), 4.34 (2H, d, J = 5.5 Hz), 3.80 (6H, s) 2 × OMe, 3.63 (2H, s) OMe.
1H NMR (300 MHz, d6- DMSO): δ 9.45 (1H, s), 8.63 (1H, t, J = 5.9 Hz), 8.51 (1H, d, J = 5.0 Hz), 8.21 (2H, d, J = 8.7 Hz), 7.99 (2H, d, J = 8.2 Hz), 7.65 (2H, d, J = 9.1 Hz), 7.37 (1H, d, J = 5.0 Hz), 6.92 (2H, d, J = 9.1 Hz), 3.99 (1H, m), 3.79 (1H, m), 3.73 (4H, m), 3.62 (1H, m), 3.30 (2H, m),* 3.04 (4H, m), 1.97-1.76 (3H, m), 1.65-1.54 (1H, m).
1H NMR (300 MHz, d6- DMSO): δ 12.44 (1H, brs), 10.90 (1H, brs), 9.46 (1H, s), 8.52 (1H, d, J = 5.0 Hz), 8.24 (2H, d, J = 8.2 Hz), 8.14 (2H, d, J = 8.7 Hz), 7.47 (3H, brd, J = 9.1 Hz),* 7.40 (1H, d, J = 5.0 Hz), 6.93 (2H, d, J = 9.1 Hz), 6.66 (1H, brs), 3.74 (4H, m), * Overlapping resonances 2H d and 1H m.
1H NMR (300 MHz, d6- DMSO): δ 9.47 (1H, s), 8.83 (1H, s), 8.53 (1H, d, J = 5.0 Hz), 8.25 (2H, d, J = 8.7 Hz), 8.01 (2H, d, J = 8.2 Hz), 7.66 (2H, d, J = 9.1 Hz), 7.39 (1H, d, J = 5.0 Hz), 6.92 (2H, d, J = 9.1 Hz), 3.73 (4H, m), 3.04 (4H, m), 1.71 (6H, s).
1HNMR (500 MHz, d6-DMSO): δ 9.58 (s, 1H), 9.33 (t, J = 5.5 Hz, 1H), 8.59 (d, J = 5.0 Hz, 1H), 8.29 (d, J = 8.3 Hz, 2H), 8.02 (d, J = 5.1 Hz, 2H), 7.63 (s, 1H), 7.47 (d, J = 5.5 Hz, 1H), 7.25 (m, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.59 (dd, J = 8.0, 2.0 Hz, 1H), 4.36 (d, J = 5.5 Hz, 2H), 3.77 (m, 4H), 3.13 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.48 (s, 1H), 9.32 (t, J = 5.4 Hz, 1H), 8.53 (d, J = 4.8 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 9.3 Hz, 2H), 7.40 (d, J = 5.1 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.41 (m, 4H), 2.70 (m, 4H).
1H NMR (300 MHz, CDCl3): δ 8.37 (1H, d, J = 5.4 Hz), 7.74 (1H, d, J = 1.5 Hz), 7.54-7.60 (3H, m), 6.98-7.07 (3H, m), 6.93 (2H, d, J = 8.7 Hz), 5.89 (1H, bs), 4.00 (3H, s), 3.88 (4H, m), 3.13 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.46 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 2H), 8.07 (brs, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.47 (brs, 1H), 7.39 (d, J = 4.8 Hz, 1H), 6.92 (d, J = 9.1 Hz, 2H), 3.73 (m, 4H), 3.04 (m, 4H).
1H NMR (500 MHz, d6- DMSO): δ 9.91 (s, 1H), 8.63 (d, J = 5.0 Hz, 1H), 8.29 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 8.5 Hz, 2H), 7.70 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 4.5 Hz, 1H), 4.36 (q, J = 7.0 Hz, 2H), 1.35 (t, J = 7.5 Hz, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.88 (s, 1H), 9.61 (t, J = 5.4 Hz, 1H), 8.61 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.7 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 5.1 Hz, 1H), 4.33 (d, J = 5.4 Hz, 2H).
1H NMR (300 MHz, CDCl3): δ 8.46 (d, J = 5.1 Hz, 1H), 8.15 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 9.3 Hz, 2H), 7.16 (bs, 1H), 7.09 (d, J = 5.4 Hz, 1H), 6.96 (d, J = 9.3 Hz, 2H), 4.00 (s, 2H), 3.90- 3.87 (m, 4 H), 3.17-3.13 (m, 4H), 3.09 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.36 (s,lH), 8.39 (s, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 7.61 (m, 2H), 7.48 (s, 2H), 6.88 (m, 2H), 3.72 (m, 4H), 3.01 (m, 4H), 2.19 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.70 (s, 1H), 9.37- 9.31 (m, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.47 (d, J = 5.1 Hz, 1H), 7.24 (d, J = 9.0 Hz, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.64-3.50 (m, 4H), 3.41 (s, 2H), 2.35 (brs, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.49 (s, 1H), 9.00 (t, J = 6.0, 1H), 8.67 (t, J = 5.4 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.05 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 4.16 (d, J = 5.4 Hz, 2H), 3.95 (d, J = 5.7 Hz, 2H), 3.78- 3.72 (m, 4H), 3.08- 3.02 (m, 4H).
1H NMR (300 MHz, CDCl3/CD3OD) δ 8.46 (d, J = 5.1 Hz, 1H), 7.92 (d, J = 1.5 Hz, 1H), 7.62 (dd, J = 1.8, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.40 (m, 1H), 7.15 (m, 2H), 7.15 (d, J = 5.4 Hz, 1H), 6.55 (m, 1H), 4.05 (s, 3H), 3.47 (s, 6H).
1H NMR (300 MHz, d6- DMSO): δ 9.44 (s, 1H), 9.38- 9.30 (m, 1H), 8.52 (d, J = 5.1, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 9.3 Hz, 2H), 7.38 (d, J = 5.1 Hz, 1H), 6.91 (d, J = 9.3 Hz, 2H), 4.69 (d, J = 4.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.68-3.50 (m, 2H), 2.82-2.68 (m, 2H), 1.93-1.74 (m, 2H), 1.58-1.39 (m, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.54 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.51 (d, J = 2.7 Hz, 1H), 8.25 (d, J = 8.1 Hz, 2H), 8.10 (d, J = 7.8 Hz, 2H), 7.98 (dd, J = 9.0 Hz, 2.7, 1H), 7.40 (d, J = 5.4 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 4.35 (q, J = 6.9 Hz, 2H), 3.74-3.68 (m, 4H), 3.40-3.33 (m, 4H), 1.34 (t, J = 7.5 Hz, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.98 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.32 (d, J = 8.1 Hz, 2H), 8.17 (s, 1H), 8.12 (d, J = 8.7 Hz, 2H), 7.97 (d, J = 9.0 Hz, 2H), 7.68 (s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 4.8 Hz, 1H), 7.09 (s, 1H), 4.36 (q, J = 6.6 Hz, 2H), 1.35 (t, J = 7.2, 3H).
1H NMR (300 MHz, CDCl3): δ 8.30 (s, 3H), 7.89 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.49 (t, J = 6.1 Hz, 1H), 4.43 (d, J = 5.9 Hz, 2H), 3.63 (d, J = 12.0 Hz, 2H), 3.55 (t, J = 5.7 Hz, 2H), 2.72-2.63 (m, 2H), 2.23 (s, 3H), 1.85 (d, J = 13.2 Hz, 2H), 1.48-1.39 (m, 2H), 1.35- 1.31 (m, 1H).
1H NMR (300 MHz, d6- DMSO): δ 9.35 (s, 1H), 8.38 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.36 (s, 1H), 7.26 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 9.3 Hz, 2H), 3.87 (s, 3H), 3.72 (m, 4H), 3.01 (m, 4H), 2.20 (s, 3H).
1H NMR (300 MHz, CD3OD): δ 8.44 (d, J = 5.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.29 (d, J = 5.4 Hz, 1H), 7.03 (d, J = 9.3 Hz, 2H), 4.53 (brs, 1H), 4.36 (s, 2H), 3.68-3.60 (m, 2H), 3.46 (d, J = 6.3 Hz, 2H), 2.73-2.64 (m, 2H), 1.92-1.82 (m, 2H), 1.68- 1.52 (m, 1H), 1.48-1.32 (m, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.78 (s, 1H), 9.37 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H), 7.76 (dd, J = 8.1, 1.8 Hz, 1H), 7.68 (d, J = 9.3 Hz, 2H), 7.35 (d, J = 5.4 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 4.06 (s, 2H), 3.98 (s, 3H), 3.74 (m, 4H), 3.04 (m, 4H).
1H NMR (300 MHz, CD3OD): δ 8.46 (d, J = 5.1 Hz, 1H), 8.25 (d, J = 8.7 Hz, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.31 (d, J = 5.4 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 4.36 (s, 2H), 3.38-3.33 (m, 4H), 3.25-3.20 (m, 4H), 2.80 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.47 (s, 1H), 8.87 (br t, J = 5.4 Hz, 1H), 8.54 (d, J = 5.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 5.1 Hz, 1H), 6.93 (d, J = 9.3 Hz, 2H), 4.32 (d, J = 5.4 Hz, 2H), 4.04 (s, 3H), 3.74 (m, 4H), 3.04 (m, 4H).
1H NMR (300 MHz, CDCl3): δ 8.31 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 9.3 Hz, 2H), 6.94 (m, 3H), 6.60 (t, J = 5.7 Hz, 1H), 4.42 (d, J = 6.1 Hz, 2H), 3.93 (m, 2H), 3.24-3.20 (m, 2H), 3.07-3.01 (m, 4H), 2.23 (s, 3H), 1.55-2.00 (m, 2H, partially obscured by grease impurity).
1H NMR (300 MHz, d6- DMSO): δ 9.33 (s, 1H), 8.87 (t, J = 5.7 Hz, 1H), 8.38 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 1.2 Hz, 1H), 7.33 (dd, J = 7.8, 1.5 Hz, 1H), 6.88 (d, J = 9.0 Hz, 2H), 4.32 (d, J = 5.7 Hz, 2H), 3.97 (s, 3H), 3.73 (m, 4H), 3.01 (m, 4H), 2.21 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 12.40 (m, 4H), 9.31 (s, 1H), 8.38 (d, J = 5.4 Hz, 1H), 8.08 (s, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 5.4 Hz, 1H), 6.90 (d, J = 9.3 Hz, 2H), 6.07 (brs, 2H), 3.74 (m, 4H), 3.04 (m, 4H), 2.80-2.63 (m, 8H).
1H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H), 8.22 (d, J = 8.7 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 9.2 Hz, 2H), 6.88 (brs, 1H), 6.45 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.8 Hz, 2H), 3.87 (t, J = 4.8 Hz, 4H), 3.87 (s, 3H), 3.12 (t, J = 4.8 Hz, 4H).
1H NMR (300 MHz, CD3OD): δ 8.56 (d, J = 5.4 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 9.0 Hz, 2H), 7.45-7.40 (m, 3H), 4.37 (s, 2H), 3.20-2.72 (m, 2H), 2.78-2.72 (m, 4H), 2.56-2.42 (m, 1H), 2.12-1.92 (m, 2H), 1.90-1.84 (m, 5H), 1.60-1.42 (m, 2H), 1.32-1.28 (brs, 1H).
1H NMR (300 MHz, d6- DMSO): δ 10.33 (s, 1H), 10.13 (t, J = 5.4 Hz, 1H), 9.35 (d, J = 5.1 Hz, 1H), 9.07 (d, J = 8.7 Hz, 2H), 8.83 (d, J = 8.4 Hz, 2H), 8.49 (d, J = 8.5 Hz, 2H), 8.22 (d, J = 5.1 Hz, 1H), 8.16- 8.02 (m, 5H), 7.73 (d, J = 9.3 Hz, 2H), 5.18 (d, J = 3.6 Hz, 2H), 5.15-5.06 (m, 1H), 4.30
1H NMR (300 MHz, d6- DMSO): δ 9.50 (s, 1H), 9.38- 9.32 (m, 1H), 8.54 (d, J = 5.1 Hz, 2H), 8.24 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.97 (dd, J = 9.0, 2.7 Hz, 1H), 7.42 (d, J = 5.4 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.76-3.68 (m, 4H), 3.40-3.35 (m, 4H).
1H NMR (300 MHz, CDCl3/CD3OD): δ 8.41 (s, 1H), 7.98 (m, 4H), 7.56 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 4.35 (s, 2H), 3.80 (t, J = 4.8 Hz, 4H), 3.08 (t, J = 4.8 Hz, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.74 (s, 1H), 9.25 (s, 1H), 8.34 (s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.39 (s, 1H), 7.28 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 4.05 (s, 2H), 3.92 (s, 3H), 3.73 (m, 4H), 3.01 (m, 4H), 2.24 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.49 (s, 1H), 9.34- 9.28 (m, 1H), 8.54 (d, J = 5.4 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.1 Hz, 1H), 6.96 (d, J = 9.0 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 3.62-3.54 (m, 4H), 3.11-3.00 (m, 4H), 2.04 (s, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.78 (brs, 1H), 8.55 (d, J = 5.4 Hz, 1H), 8.22 (d, J = 8.7 Hz, 2H), 7.81 (brd, J = 8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.48 (d, J = .8 Hz, 2H), 7.44 (d, J = 5.1 Hz, 1H), 7.29 (brd, J = 8.1 Hz, 2H), 7.11 (ap. d, J = 7.8 Hz, 2H), 4.58 (d, J = 2.4 Hz, 2H), 3.86 (m, 4H), 3.41 (t, J = 2.4 Hz, 1H), 3.34 (brm, 4H), 3.13 (s, 3H), 2.28 (s, 3H).
1H NMR (300 MHz, CD3OD): δ 8.27 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 9.1 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.34 (s, 2H), 3.78-3.68 (m, 1H), 3.45-3.41 (m, 2H), 2.80-2.75 (m, 2H), 2.21 (s, 3H), 1.93-1.91 (m, 2H), 1.65-1.62 (m, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.51 (s, 1H), 9.36- 9.30 (m, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 6.93 (d, J = 10.2 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 4.32-4.26 (m, 1H), 3.00-1.35 (m, 9H).
1H NMR (300 MHz, d6- DMSO): δ 10.11 (1H, brs), 9.64 (1H, s), 9.27 (1H, brd, J = 5.0 Hz), 8.48 (1H d, J = 5.0 Hz), 8.33 (1H, d, J = 2.8 Hz), 8.20 (2H, d, J = 8.9 Hz), 7.85 (1H, dd, J = 2.8, J = 8.7 Hz), 7.35 (3H, m), 7.22 (1H, d, J = 8.9 Hz), 3.75 (4H, m), 3.07 (3H, s), 2.87 (7H, m).
1H NMR (300 MHz, CDCl3): δ 8.97 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.9 Hz, 2H), 7.36 (brs, 1H), 6.92 (d, J = 9.0 Hz, 2H), 6.51 (t, J = 5.1 Hz, 1H), 4.41 (d, J = 5.8 Hz, 2H), 4.18 (q, J = 7.2 Hz, 2H), 3.87 (t, J = 4.8 Hz, 4H), 3.14 (t, J = 4.8 Hz, 4H), 1.16 (t, J = 7.1 Hz, 3H)
1H NMR (300 MHz, d6- DMSO): δ 9.33 (t, J = 5.4 Hz, 1H), 8.65 (d, J = 5.7 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.32- 8.29 (m, 3H), 8.12 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.60-7.53 (m, 3H), 4.36 (d, J = 5.1 Hz, 2H), 3.96 (s, 3H), 3.80-3.67 (m, 1H), 2.81- 2.77 (m, 2H), 2.18 (s, 3H), 2.00-1.90 (m, 2H), 1.80 (m, 2H), 1.67-1.53 (m, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.23 (t, J = 5.4 Hz, 1H), 8.86 (s, 1H), 8.18 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.03-6.99 (m, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.34 (d, J = 5.4 Hz, 2H), 3.76-3.72 (m, 4H), 3.05-3.01 (m, 4H).
1H NMR (300 MHz, CD3OD/d6- DMSO): δ 8.58 (s, 1H), 8.01 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 9.2 Hz, 2H), 4.35 (s, 2H), 3.80 (t, J = 4.8 Hz, 4H), 3.08 (t, J = 4.8 Hz, 4H)
1H NMR (300 MHz, d6- DMSO): δ 9.71 (s, 1H), 9.38- 9.33 (m, 1H), 8.58 (d, J = 5.4 Hz, 1H), 8.28 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.77 (dd, J = 8.4 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.47 (d, J = 4.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.70-3.63 (m, 4H), 3.12- 2.98 (m, 5H), 2.82 (s, 3H), 2.80-2.66 (m, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.79 (s, 1H), 9.53 (s, 1H), 9.38-9.33 (m, 1H), 8.61 (d, J = 5.1 Hz, 1H), 8.45 (d, J = 2.7 Hz, 1H), 8.35 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.85 (dd, J = 8.7, J = 2.7 Hz, 1H), 7.50 (d, J = 5.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 3.80- 3.73 (m, 4H), 2.94-2.88 (m, 4H), 1.44 (s, 9H).
1H NMR (300 MHz, d6- DMSO): δ 9.78 (s, 1H), 9.60- 9.45 (m, 1H), 9.37-9.32 (m, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.41 (d, J = 8.4, 2.7 Hz, 1H), 8.35 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.83 (dd, J = 2.7, 8.7 Hz, 1H), 7.50 (d, J = 5.1 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.80-3.73 (m, 4H), 3.44- 3.30 (m, 2H), 2.92-2.88 (m, 4H), 1.20 (t, J = 7.2 Hz, 3H).
1H NMR (300 MHz, d6- DMSO): δ 9.77 (brs, 1H), 9.33 (t, J = 5.4 Hz, 1H), 8.60 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.7 Hz, 2H), 7.78 (dd, J = 15.6, 2.4 Hz, 1H), 7.47-7.53 (m, 2H), 7.06- 6.99 (m, 1H), 4.36 (d, J = 5.7 Hz, 2H), 3.76-3.72 (m, 4H), 2.98-2.94 (m, 4H).
1H NMR (300 MHz, CD3OD/d6- DMSO): δ 8.33 (d, J = 5.4 Hz, 1H), 8.18 (m, 1H), 7.99-7.84 (m, 2H), 7.52 (d, J = 9.2 Hz, 2H), 7.16 (d, J = 5.1 Hz, 1H), 6.88 (d, J = 9.1 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.01 (t, J = 4.8 Hz, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.43 (s, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.20 (s, 1H), 8.16 (s, 2H), 7.64 (d, J = 9.1 Hz, 2H), 7.36 (d, J = 5.1 Hz, 1H), 6.91 (d, J = 9.1 Hz, 2H), 4.04 (brs, 1H), 3.74 (t, J = 4.8 Hz, 4H), 3.45 (m, 2H, obscured by water signal), 3.04 (t, J = 4.8 Hz, 4H).
1H NMR (300 MHz, d6- DMSO): δ 10.02 (s, 1H), 9.40- 9.32 (m, 1H), 8.64 (d, J = 5.1 Hz, 1H), 8.46 (brs, 1H), 8.30 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 8.1 Hz, 2H), 7.96 (dd, J = 9.3, 1.8 Hz, 1H), 7.59-7.54 (m, 2H), 4.36 (d, J = 5.4 Hz, 2H), 2.76-2.66 (m, 4H), 2.85-2.80 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.89 (1H, t, J = 5.2 Hz), 9.77 (1H, s), 8.57 (1H, d, J = 5.5 Hz), 8.48 (1H, d, J = 2.7 Hz), 8.32 (2H, d, J = 9.1 Hz), 7.91 (1H, dd, J = 2.7, 8.7 Hz), 7.62 (2H, d, J = 8.7 Hz), 7.45 (1H, d, J = 5.5 Hz), 7.33 (1H, d, J = 8.7 Hz), 4.96 (2H, s), 3.80 (4H, m), 3.45 (2H, m), 3.20 (3H, s), 2..88 (4H, m), 2.45 (2H, m), 2.21 (6H, s).
1H NMR (300 MHz, d6- DMSO): δ 9.97 (1H, s), 8.62 (1H, d, J = 5.0 Hz), 8.36 (1H, d, J = 2.2 Hz), 8.26 (2H, d, J = 8.7 Hz), 8.02 (1H, dd, J = 2.3, J = 8.7 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.54 (1H, d, J = 8.7 Hz), 7.51 (1H, d, J = 5.1 Hz), 4.97 (2H, s), 3.69 (4H, m), 3.21 (3H, s), 2.82 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.73 (s, 1H), 9.34 (t, J = 4.8 Hz, 1H), 8.60 (d, J = 5.1 Hz, 1H), 8.30 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.91 (brs, 1H), 7.69 (brs, J = 4.3 Hz, 1H), 7.49 (d, J = 5.4 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 4.46 (s, 2H), 4.35 (d, J = 5.1 Hz,
1H NMR (300 MHz, d6- DMSO): δ 10.16 (s, 1H), 9.50 (s, 1H), 8.54 (d, J = 5.0 Hz, 1H), 8.52 (d, J = 2.5 Hz, 1H), 8.42 (dd, J = 8.0, 2.0 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 5.0 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 3.98 (s, 2H), 3.74 (t, J = 4.5 Hz, 4H), 3.04 (t, J = 5.0 Hz, 4H).
1H NMR (300 MHz, d6- DMSO): δ 12.92 (s, 1H), 9.70 (s, 1H), 9.35 (t, J = 5.6 Hz, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.30 (d, J = 8.4 Hz, 3H), 8.04 (d, J = 8.4 Hz, 3H), 7.64 (m, 1H), 7.47 (m, 2H), 4.36 (d, J = 5.7 Hz, 2H).
1H NMR (300 MHz, d6- DMSO): δ 9.49 (s, 1H), 9.45 (s, 1H), 8.52 (d, J = 5 Hz, 1H), 8.24 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 9 Hz, 2H), 7.39 (d, J = 5 Hz, 1H), 6.92 (d, J = 9 Hz, 2H), 3.74 (m, 4H), 3.04 (m, 4H), 1.58 (m, 2H), 1.31 (m, 2H).
1H NMR (300 MHz, CDCl3): δ 8.49 (1H, d, J = 5.5 Hz), 8.17 (2H, d, J = 7.8 Hz), 7.89 (2H, d, J = 8.2 Hz), 7.75 (1H, d, J = 2.6 Hz), 7.65 (1H, dd, J = 8.7, 2.3 Hz), 7.23 (1H, brs), 7.15 (1H, d, J = 5.5 Hz), 7.12 (1H, d, J = 8.5 Hz), 6.65 (1H, brs), 6.08- 5.95 (1H, m), 5.37-5.30 (1H, m), 5.23-5.19 (1H, m), 4.65 (2H, s), 4.42 (2H, d, J = 6.1 Hz), 4.12 (2H, d, J = 5.3 Hz), 3.84 (4H, t, J = 4.4 Hz), 2.92
1H NMR (300 MHz, d6- DMSO): δ 9.54 (s, 1H), 9.32 (m, 1H), 8.54 (d, J = 5.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 5.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 4.31 (m, 3H), 2.69 (m, 2H), 2.32 (m, 2H), 2.14 (m, 2H), 1.93 (m, 2H), 1.58 (m,
1H NMR (300 MHz, d6- DMSO): δ 9.74 (1H, s), 8.57 (1H, d, J = 5.0 Hz), 8.24 (2H, d, J = 8.7 Hz), 7.75 (1H, dd, J = 2.2, 15.5 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.52 (1H, brdd, J = 2.0, 8.7 Hz), 7.43 (1H, d, J = 5.0 Hz), 7.02 (1H, dd, J = 8.7, 9.1 Hz), 4.96 (2H, s), 3.73 (4H, m), 3.21 (3H, s), 2.94 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.88 (1H, s), 8.60 (1H, d, J = 5.5 Hz), 8.23 (2H, d, J = 8.7 Hz), 8.17 (1H, d, J = 2.7 Hz), 8.02 (1H, dd, J = 2.7, 9.1 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.48 (1H, d, J = 5.5 Hz), 7.21 (1H, d, J = 9.1 Hz), 4.96 (2H, s), 3.75 (4H, m), 3.20 (3H, s), 3.06 (4H, m).
1H NMR (CDCl3/CD3OD, 300 MHz): δ 8.47 (d, J = 5.1 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 1.5 Hz, 1H), 7.57 (dd, 1.5, J = 8.4 Hz, 1H), 7.44 (dd, J = 2.4, 2.1 Hz, 1H), 7.19 (dd, J = 7.8, 7.8 Hz, 1H), 7.15 (d, J = 5.4 Hz, 1H), 7.05 (ddd, J = 0.9, 2.1, 8.1 Hz, 1H), 6.56 (ddd, J = 0.9, 2.4, 8.1Hz, 1H), 4.36 (t, J = 6.7 Hz, 2H), 1.86 (m, 2H), 1.07 (t, J = 5.7 Hz, 3H).
1H NMR (300 MHz, d6- acetone): δ 8.92 (d, J = 2.1 Hz, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.4 (d, J = 5.3 Hz, 1H), 8.17 (dd, J = 8.9, 2.2 Hz, 1H), 8.09 (dd, J = 9.1, 2.8 Hz, 1H), 7.25 (d, J = 5.3 Hz, 1H), 7.18 (d, J = 8.9 Hz, 1H), 6.83 (d, J = 9.1 Hz, 1H), 3.75 (m, 4H), 3.42 (m, 4H), 3.22 (bs, 2H).
1H NMR (300 MHz, d6- DMSO): δ 10.53 (s, 1H), 9.36 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 8.14 (d, J = 8.7 Hz, 2H), 7.69 (m, 4H), 7.28 (d, J = 5.4 Hz, 1H), 6.94 (d, J = 9.1 Hz, 2H), 3.96 (s, 2H), 3.74 (m, 4H), 3.05 (m, 4H)
1H NMR (300 MHz, d6- DMSO): δ 17.23 (1H, s) CO2H, 9.99 (1H, s), 8.74 (1H, d, J = 2.7 Hz), 8.62 (1H, d, J = 5.0 Hz), 8.33 (2H, d, J = 8.7 Hz), 8.01 (1H, dd, J = 2.7, 8.7 Hz), 7.69 (1H, d, J = 9.1 Hz), 7.63 (2H, d, J = 8.7 Hz), 7.52 (1H, d, J = 5.5 Hz), 4.97 (2H, s), 3.81 (4H, m), 3.21 (3H, s), 3.06 (4H, m).
1H NMR (300 MHz, d6- DMSO): δ 9.51 (s, 1H), 9.34 (t, J = 5.5, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 6.90 (d, J = 9.0 Hz, 2H), 4.34 (d, J = 5.4 Hz, 2H), 3.98 (t, J = 6.5 Hz, 2H), 2.54 (m, 6H), 1.84 (m,
1H NMR (300 MHz, d6- DMSO): δ 9.53 (s, 1H), 9.32 (t, J = 5.4 Hz, 1H), 8.55 (d, J = 5.4 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H), 7.42 (d, J = 5.4 Hz, 1H), 6.92 (d, J = 9.3 Hz, 2H), 4.35 (d, J = 5.4 Hz, 2H), 4.06 (t, J = 5.7 Hz, 2H), 3.58 (m, 4H), 2.69 (t, J = 5.8 Hz, 2H), 2.48 (m, 4H, partially obscured by DMSO signal).
1H NMR (300 MHz, d6- DMSO): δ 9.52 (s, 1H), 9.32 (t, J = 5.6 Hz, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 5.4 Hz, 1H), 7.02 (d, J = 5.4 Hz, 2H), 4.35 (d, J = 5.1 Hz, 2H), 3.70 (m, 4H), 3.14 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.73 (s, 1H), 9.33 (t, J = 5.4 Hz, 1H), 8.60 (d, J = 5.1 Hz, 1H), 8.29 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 5.1 Hz, 1H), 7.27 (d, J = 8.7 Hz, 2H), 4.36 (d, J = 5.4 Hz, 2H), 3.62 (s, 2H), 3.10 (m, 4H), 2.88 (m, 4H).
1H NMR (300 MHz, d6- DMSO): δ 9.46 (s, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.23 (d, J = 8.7 Hz, 2H), 7.65 (m, 4H), 7.37 (d, J = 5.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 4.56 (brs, 2H), 3.74 (m, 4H), 3.04 (m, 7H).
1H NMR (300 MHz, CDCl3): δ 8.31 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 6.96 (brs, 1H), 6.91 (d, J = 9.2 Hz, 2H), 6.56 (t, J = 5.7 Hz, 1H), 4.42 (d, J = 5.8 Hz, 2H), 3.86 (t, J = 4.8 Hz, 4H), 3.11 (t, J = 4.8 Hz, 4H), 2.23 (s, 3H).
1H NMR (300 MHz, CDCl3): δ 8.35 (d, J = 3.3 Hz, 1H), 8.22 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.9 Hz, 2H), 7.51 (d, J = 9.1 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 6.45-6.44 (m, 1H), 4.43 (d, J = 5.7 Hz, 2H), 3.88 (t, J = 4.7 Hz, 4H), 3.13 (t, J = 4.8 Hz, 4H).
The terms “C1-6alkyl” and “C1-4alkyl” refers to straight chain or branched chain hydrocarbon groups having from 1 to 6 carbon atoms. Examples include ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl.
The terms “C1-6alkylene” and “C1-4alkylene” are the divalent equivalents of “C1-6alkyl” and “C1-4alkyl”.
The term “C2-4alkenyl” refers to straight chain or branched chain hydrocarbon groups having at least one double bond of either E or Z stereochemistry where applicable and 2 to 4 carbon atoms. Examples include vinyl, 1-propenyl, 1-and 2-butenyl and 2-methyl-2-propenyl.
The term “aryl” refers to single, polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenxanthracenyl and phenanthrenyl.
The term “unsaturated N-containing 5 or 6-membered heterocyclyl” refers to unsaturated, cyclic hydrocarbon groups containing at least one nitrogen.
Suitable N-containing heterocyclic groups include unsaturated 5 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; unsaturated 5 or 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; and unsaturated 5 or 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl.
The term “halogen” refers to fluorine, chlorine, bromine and iodine.
The term “substituted” refers to a group that is substituted with one or more groups selected from C1-6 alkyl, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylaryl, aryl, heterocycylyl, halo, haloC1-6alkyl, haloC3-6cycloalkyl, haloC2-6alkenyl,
haloC2-6alkynyl, haloaryl, haloheterocycylyl, hydroxy, C1-6 alkoxy, C2-6alkenyloxy, C2-6alkynyloxy, aryloxy, heterocyclyloxy, carboxy, haloC1-6alkoxy,
haloC2-6alkenyloxy, haloC2-6alkynyloxy, haloaryloxy, nitro, nitroC1-6, alkyl, nitroC2-6alkenyl, nitroaryl, nitroheterocyclyl, azido, amino, C1-6alkylamino,
C2-6alkenylamino, C2-6alkynylamino, arylamino, heterocyclamino acyl, C1-6alkylacyl, C2-6alkenylacyl, C2-6alkynylacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy, aldehydo, C1-6alkylsulphonyl, arylsulphonyl, C1-6alkylsulphonylamino, arylsulphonylamino,
C1-6alkylsulphonyloxy, arylsulphonyloxy, C1-6alkylsulphenyl, C2-6alklysulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, C1-6alkylthio, arylthio, acylthio, cyano and the like. Preferred substituents are selected from the group consisting of C1-4 alkyl, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylaryl, aryl, heterocycylyl, halo, haloaryl, haloheterocycylyl, hydroxy, C1-4 alkoxy, aryloxy, carboxy, amino,
C1-6alkylacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy, C1-6alkylsulphenyl, arylsulphonyl and cyano.
The compounds of the invention may also be prepared as salts which are pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, trihalomethanesulfonic, toluenesulfonic, benzenesulfonic, isethionic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, valeric and orotic acids. Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.
The salts may be formed by conventional means, such as by reacting the free base form of the compound with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.
Where a compound possesses a chiral center the compound can be used as a purified enantiomer or diastereomer, or as a mixture of any ratio of stereoisomers. It is however preferred that the mixture comprises at least 70%, 80%, 90%, 95%, 97.5% or 99% of the preferred isomer, where the preferred isomer gives the desired level of potency and selectivity.
This invention also encompasses prodrugs of the compounds of formula I. The invention also encompasses methods of treating disorders that can be treated by the inhibition of protein kinases, such as JAK comprising administering drugs or prodrugs of compounds of the invention. For example, compounds of formula I having free amino, amido, hydroxy or carboxylic acid groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy and carboxylic acid groups of compounds of the invention. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methioine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of compounds of the present invention through the carbonyl carbon prodrug sidechain. Prodrugs also include phosphate derivatives of compounds (such as acids, salts of acids, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula I. Prodrugs may also include N-oxides, and S-oxides of appropriate nitrogen and sulfur atoms in formula I.
Process
Compounds of the general formula I are generally prepared from a dichloropyrimidine.
The first step of the process typically begins with a cross-coupling reaction between a 2,4 dichloropyrimidine and a suitably functionalised coupling partner. Alternately the dichloropyrimidine may be converted to a diiodopyrimidine, which is then coupled with a suitably functionalised coupling partner. Typical coupling partners are organoboronic acids or esters (Suzuki coupling: see for example Miyaura, N. and Suzuki, Chem Rev. 1995, 95 2457), organostannanes (Stille coupling: see for example Stille, J. K., Angew. Chem., Int. Ed. Engl., 1986, 25, 508), Grignard reagents (Kumada coupling: Kumada, M.; Tamao, K.; Sumitani, K. Org. Synth. 1988, Coll. Vol. 6, 407.) or organozinc species (Negishi coupling: Negishi, E.; J. Organomet. Chem. 2002, 653, 34). The Suzuki coupling is the preferred coupling method and is typically performed in a solvent such as DME, THF, DMF, ethanol, propanol, toluene, acetonitrile or 1,4-dioxane, with or without added water, in the presence of a base such as sodium or potassium carbonate, lithium hydroxide, caesium carbonate, sodium hydroxide, potassium fluoride or potassium phosphate. The reaction may be carried out at elevated temperatures and the palladium catalyst employed may be selected from Pd(PPh3)4, Pd(OAc)2, [PdCl2(dppf)], Pd2(dba)3/P(t-Bu)3.
The second step of the process involves a nucleophilic aromatic substitution reaction of the derived above with a suitably substituted aniline. The nucleophilic aromatic substitution is typically carried out by addition of the aniline to monohalo heterocyclic intermediate obtained from the first reaction in a solvent such as ethanol, n-propanol, isopropanol, tert-butanol, dioxane, THF, DMF, toluene or xylene. The reaction is typically performed at elevated temperature in the presence of an acid such as HCl or p-toluenesulfonic acid or in the presence of base such as a non-nucleophilic base such as triethylamine or diisopropylethylamine, or an inorganic base such as potassium carbonate or sodium carbonate.
Alternatively, the aniline substituent may be introduced through a transition metal catalysed amination reaction. Typical catalysts for such transformations include Pd(OAc)2/P(t-Bu)3, Pd2(dba)3/BINAP and Pd(OAc)2/BINAP. These reactions are typically carried out in solvents such as toluene or dioxane, in the presence of bases such as caesium carbonate or sodium or potassium tert-butoxide at temperatures ranging from room temperature to reflux (e.g., Hartwig, J. F., Angew. Chem. Int. Ed. 1998, 37, 2046).
The anilines employed in the first step of the synthesis of these compounds are obtained commercially or are prepared using methods well known to those skilled in the art.
The products formed from either reaction step may be further derivatised using techniques known to those skilled in the art. Alternatively, derivatisation of the mono-halo intermediate may be undertaken prior to displacement of the halo substituent. Those skilled in the art will appreciate that the order of the reactions described for the syntheses above may be changed in certain circumstances and that certain functionalities may need to be derivatised (i.e., protected) in certain instances for the reactions described above to proceed with reasonable yield and efficiency. The types of protecting functionality are well-known to those skilled in the art and are described for example in Greene (Greene, T., Wuts, P. (1999) Protective Groups in Organic Synthesis. Wiley-Interscience; 3rd edition).
The leaving group in the compound of formula II which is an intermediate used in the process of the present invention may be any suitable known type such as those disclosed in J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure” 4th Edition, pp 352-357, John Wiley & Sons, New York, 1992 which is incorporated herein by reference. Preferably, the leaving group is halogen, more preferably chlorine or iodine.
JAK Inhibition
The compounds of formula I have activity against protein kinases, particularly the JAK kinases and most particularly are active against JAK2. A JAK2 inhibitor is any compound that selectively inhibits the activity of JAK2. One activity of JAK2 is to phosphorylate a STAT protein. Therefore an example of an effect of a JAK2 inhibitor is to decrease the phosphorylation of one or more STAT proteins. The inhibitor may inhibit the phosphorylated form of JAK2 or the non-phosphorylated form of JAK2.
The present invention also provides the use of kinase inhibitors such as JAK kinase inhibitors, in particular JAK2 inhibitors.
Pharmaceutical Compositions
The present invention provides pharmaceutical compositions comprising at least one of the compounds of the formula I and a pharmaceutically acceptable carrier. The carrier must be “pharmaceutically acceptable” means that it is compatible with the other ingredients of the composition and is not deleterious to a subject. The compositions of the present invention may contain other therapeutic agents as described below, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).
The compounds of the invention may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally such as by inhalation spray or insufflation; topically, such as in the form of a cream or ointment ocularly I the form of a solution or suspension; vaginally in the form of pessaries, tampons or creams; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. The compounds may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
The pharmaceutical compositions for the administration of the compounds of the invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. These methods generally include the step of bringing the compound of formula I into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the compound of formula I into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
The pharmaceutical compositions containing the compound of formula I may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents such as sweetening agents, flavouring agents, colouring agents and preserving agents, e.g., to provide pharmaceutically stable and palatable preparations. Tablets contain the compound of formula I in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard gelatin capsules wherein the compound of formula I is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the compound of formula I is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the compound of formula I in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound of formula I in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable formulations.
For administration to the respiratory tract, including intranasal administration, the active compound may be administered by any of the methods and formulations employed in the art for administration to the respiratory tract.
Thus in general the active compound may be administered in the form of a solution or a suspension or as a dry powder.
Solutions and suspensions will generally be aqueous, for example prepared from water alone (for example sterile or pyrogen-free water) or water and a physiologically acceptable co-solvent (for example ethanol, propylene glycol or polyethylene glycols such as PEG 400).
Such solutions or suspensions may additionally contain other excipients for example preservatives (such as benzalkonium chloride), solubilising agents/surfactants such as polysorbates (e.g., Tween® 80, Span 80, benzalkonium chloride), buffering agents, isotonicity-adjusting agents (for example sodium chloride), absorption enhancers and viscosity enhancers. Suspensions may additionally contain suspending agents (for example microcrystalline cellulose and carboxymethyl cellulose sodium).
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case a means of dose metering is desirably provided. In the case of a dropper or pipette this may be achieved by the subject administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomising spray pump.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the compound is provided in a pressurised pack with a suitable propellant, such as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of active compound may be controlled by provision of a metered valve.
Alternatively the active compound may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form, for example in capsules or cartridges of e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
In formulations intended for administration to the respiratory tract, including intranasal formulations, the active compound will generally have a small particle size, for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.
When desired, formulations adapted to give sustained release of the active compound may be employed.
The active compound may be administered by oral inhalation as a free-flow powder via a “Diskhaler” (trade mark of Glaxo Group Ltd) or a meter dose aerosol inhaler.
The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.
Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. (For purposes of this application, topical application shall include mouthwashes and gargles.)
For application to the eye, the active compound may be in the form of a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle. Additives, for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorohexidine and thickening agents such as hypromellose may also be included.
The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and phospha-tidyl cholines, both natural and synthetic. Methods to form liposomes are known in the art.
Efficacy of this class of compounds may be applicable to drug eluting stents. Potential applications of drug eluting stents with these compounds include pulmonary artery stenosis, pulmonary vein stenosis, as well as coronary artery stenosis. Drug eluting stents may also be used in saphenous vein grafts or arterial grafts or conduits. Drug eluting stents that release this class of compounds may also be applicable for treating stenoses of the aorta or peripheral arteries, such as the iliac artery, the femoral artery or the popliteal artery. The compound may be bound to the drug eluting stent by any of various methods known in the field. Examples of such methods include polymers, phosphoryl choline, and ceramics. The compound may also be impregnated into a bioabsorbable stent.
The active compounds may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:
oral administration, external application, for example drenches (e.g., aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g., as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;
topical applications, e.g., as a cream, ointment or spray applied to the skin; or
rectally or intravaginally, e.g., as a pessary, cream or foam.
The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Examples of other therapeutic agents include the following: endothelin receptor antagonists (e.g., ambrisentan, bosentan, sitaxsentan), PDE-V inhibitors (e.g., sildenafil, tadalafil, vardenafil), Calcium channel blockers (e.g., amlodipine, felodipine, varepamil, diltiazem, menthol), prostacyclin, treprostinil, iloprost, beraprost, nitric oxide, oxygen, heparin, warfarin, diuretics, digoxin, cyclosporins (e.g., cyclosporin A), CTLA4 Ig, antibodies such as ICAM 3, anti IL 2 receptor (Anti Tac), anti CD45RB, anti CD2, anti CD3 (OKT 3), anti CD4, anti CD80, anti CD86, agents blocking the interaction between CD40 and gp39, such as antibodies specific for CD40 and/or gp39 (i.e., CD154), fusion proteins constructed from CD40 and gp39 (CD401g and CD8gp39), inhibitors, such as nuclear translocation inhibitors, of NF kappa B function, such as deoxyspergualin (DSG), cholesterol biosynthesis inhibitors such as HMG CoA reductase inhibitors (lovastatin and simvastatin), non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin, acetaminophen, leflunomide, deoxyspergualin, cyclooxygenase inhibitors such as celecoxib, steroids such as prednisolone or dexamethasone, gold compounds, beta-agonists such as salbutamol, LABA's such as salmeterol, leukotriene antagonists such as montelukast, antiproliferative agents such as methotrexate, FK506 (tacrolimus, Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprine, VP-16, etoposide, fludarabine, doxorubin, adriamycin, amsacrine, camptothecin, cytarabine, gemcitabine, fluorodeoxyuridine, melphalan and cyclophosphamide, antimetabolites such as methotrexate, topoisomerase inhibitors such as camptothecin, DNA alkylators such as cisplatin, kinase inhibitors such as sorafenib, microtubule poisons such as paclitaxel, TNF-inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, hydroxy urea and rapamycin (sirolimus or Rapamune) or derivatives thereof.
When other therapeutic agents are employed in combination with the compounds of the present invention they may be used for example in amounts as noted in the Physician Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.
Methods of Treatment
The compounds of formula I may be used in the treatment of kinase associated diseases including JAK kinase associated diseases such as immunological and inflammatory diseases including organ transplants; hyperproliferative diseases including cancer and myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases.
Generally, the term “treatment” means affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.
The term “subject” refers to any animal having a disease which requires treatment with the compound of formula I.
In addition to primates, such as humans, a variety of other mammals can be treated using the compounds, compositions and methods of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. However, the invention can also be practiced in other species, such as avian species (e.g., chickens).
The term “administering” should be understood to mean providing a compound of the invention to a subject in need of treatment.
The term “kinase associated diseases” refers to a disorder or disorders that directly or indirectly result from or are aggravated by aberrant kinase activity, in particular JAK activity and/or which are alleviated by inhibition of one or more of these kinase enzymes.
In a preferred embodiment the kinase associated disease state involves one or more of the JAK kinases, JAK1, JAK2, JAK3 or TYK2. In a particularly preferred embodiment, the disease involves JAK2 kinase. Such diseases include, but are not limited to, those listed in the Table below.
The term “immunological and inflammatory disease” refers to an immunological, inflammatory or autoimmune disease, including but not limited to rheumatoid arthritis, polyarthritis, rheumatoid spondylitis, osteoarthritis, gout, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, mucous colitis, ulcerative colitis, diabrotic colitis, Crohn's disease, autoimmune thyroid disorders, gastritis, esophagitis, hepatitis, pancreatitis, nephritis, psoriasis, eczema, acne vulgaris, dermatitis, hives, multiple sclerosis, Alzheimer's disease, Motor Neurone Disease (Lou Gehrig's disease), Paget's disease, sepsis, conjunctivitis, neranl catarrh, chronic arthrorheumatism, systemic inflammatory response syndrome (SIRS), polymyositis, dermatomyositis (DM), Polaritis nodoa (PN), mixed connective tissue disorder (MCTD), Sjögren's syndrome, Crouzon syndrome, achondroplasia, systemic lupus erythematosus, scleroderma, vasculitis, thanatophoric dysplasia, insulin resistance, Type I diabetes and complications from diabetes and metabolic syndrome.
The term “hyperproliferative diseases” includes cancer and myeloproliferative disease states such as cellular-proliferative disease states, including but not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hanlartoma, inesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinorna, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilms tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostrate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfrorna (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis defornians), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, SertoliLeydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma]), fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; Adrenal glands: neuroblastoma; and Myleoproliferative diseases such as polycythemia vera (PV), primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), agnoneic myeloid metaplasia (AMM), also referred to as idiopathic myelofibrosis (IMF), chronic myelogenous leukemia (CML), systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodisplastic syndrome (MDS) and systemic mast cell disease (SMCD).
The term “vascular diseases” refers to diseases including but not limited to cardiovascular diseases, hypertension, hypertrophy, hypercholesterolemia, hyperlipidemia, thrombotic disorders, stroke, Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine, peripheral arterial disease, heart failure, restenosis, atherosclerosis, left ventricular hypertrophy, myocardial infarction, ischemic diseases of heart, kidney, liver and brain, and pulmonary arterial hypertension.
Preferred diseases for JAK2 selective inhibitors include immunological and inflammatory diseases such as auto-immune diseases for example atopic dermatitis, asthma, rheumatoid arthritis, Crohn's disease, psoriasis, Crouzon syndrome, achondroplasia, systemic lupus erythematosus, scleroderma, mixed connective tissue disease, vasculitis, thanatophoric dysplasia and diabetes; hyperproliferative disorders such as cancer for example prostate cancer, colon cancer, breast cancer, liver cancer such as hepatoma, lung cancer, head and neck cancer such as glioma, skin cancer such as metastatic melanoma, leukemia, lymphoma, multiple myeloma and myeloproliferative diseases such as polycythemia vera (PV), myelofibrosis, thrombocythemia, essential thrombocythemia (ET), agnoneic myeloid metaplasia (AMM), also referred to as idiopathic myelofibrosis (IMF) and chronic myelogenous leukemia (CML); and vascular diseases such as hypertension, hypertrophy, stroke, Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine, peripheral arterial disease, heart failure, restenosis, atherosclerosis and pulmonary arterial hypertension.
Dosages
The term “therapeutically effective amount” refers to the amount of the compound of formula I and II that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
In the treatment or prevention of conditions which require kinase inhibition an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient. The dosage may be selected, for example to any dose within any of these ranges, for therapeutic efficacy and/or symptomatic adjustment of the dosage to the patient to be treated. The compounds will preferably be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
In order to exemplify the nature of the present invention such that it may be more clearly understood, the following non-limiting examples are provided.
Compound Synthesis
The compounds of the invention may be prepared by methods well known to those skilled in the art, and as described in the synthetic and experimental procedures shown below for selected compounds.
A mixture of 4-ethoxycarbonylphenyl boronic acid (23.11 g, 119 mmol), 2,4-dichloropyrimidine (16.90 g, 113 mmol), toluene (230 mL) and aqueous sodium carbonate (2 M, 56 mL) was stirred vigorously and nitrogen was bubbled through the suspension for 15 minutes. Tetrakis(triphenylphosphine)palladium[0] (2.61 g, 2.26 mmol) was added. Nitrogen was bubbled through for another 10 min., the mixture was heated to 100° C., then at 75° C. overnight. The mixture was cooled, diluted with ethyl acetate (200 mL), water (100 mL) was added and the layers were separated. The aqueous layer was extracted with ethyl acetate (100 ml) and the two organic extracts were combined. The organics were washed with brine, filtered through sodium sulfate, concentrated, and the resultant solid was triturated with methanol (100 mL) and filtered. The solids were washed with methanol (2×30 mL) and air dried. This material was dissolved in acetonitrile (150 mL) and dichloromethane (200 mL), stirred with MP.TMT Pd-scavenging resin (Agronaut part number 800471) (7.5 g) over 2 days. The solution was filtered, the solids were washed with dichloromethane (2×100 mL), and the filtrate concentrated to give ethyl 4-(2-chloropyrimidin-4-yl)benzoate as an off-white solid (17.73 g, 60%)—additional washing with dichloromethane yielded a further 1.38 g and 0.5 g of product. 1H NMR (300 MHz, d6-DMSO) δ 8.89 (1H, d, J=5.0 Hz); 8.32 (2H, d, J=8.7 Hz); 8.22 (1H, d, J=5.5 Hz); 8.12 (2H, d, J=8.7 Hz); 4.35 (2H, q, J=7.1 Hz); 1.34 (3H, t, J=7.1 Hz); LC-ESI-MS (method B): rt 7.3 min.; m/z 263.0/265.0 [M+H]+.
A mixture of ethyl 4-(2-chloropyrimidin-4-yl)benzoate (26.15 g, 99.7 mmol) and 4-morpholinoaniline (23.10 g, 129.6 mmol) was suspended in 1,4-dioxane (250 mL). p-Toluenesulfonic acid monohydrate (17.07 g, 89.73 mmol) was added. The mixture was heated at reflux for 40 h., cooled to ambient temperature, concentrated then the residue was partitioned between ethyl acetate and 1:1 saturated sodium bicarbonate/water (1 L total). The organic phase was washed with water (2×100 mL) and concentrated. The aqueous phase was extracted with dichloromethane (3×200 mL). The material which precipitated during this workup was collected by filtration and set aside. The liquid organics were combined, concentrated, triturated with methanol (200 mL) and filtered to yield additional yellow solid. The solids were combined, suspended in methanol (500 mL), allowed to stand overnight then sonicated and filtered. The solids were washed with methanol (2×50 mL) to give, after drying, ethyl 4-(2-(4-morphonlinophenylamino)pyrimidin-4-yl)benzoate (35.39 g, 88%). 1H NMR (300 MHz, d6-DMSO) δ 9.49 (1H, s); 8.54 (1H, d, J=5.0 Hz); 8.27 (2H, d, J=8.7 Hz); 8.10 (2H, d, J=8.7 Hz), 7.66 (2H, d, J=9.1 Hz); 7.38 (1H, d, J=5.0 Hz); 6.93 (2H, d, J=8.7 Hz); 4.35 (2H, q, J=6.9 Hz), 3.73 (4H, m); 3.04 (4H, m); 1.34 (3H, t, J=6.9 Hz); LC-ESI-MS (method B): rt 7.5 min.; m/z 404.1 [M+H]+.
A solution of ethyl 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoate (35.39 g, 87.6 mmol) in 3:1 methanol/tetrahydrofuran (350 mL) was treated with lithium hydroxide (4.41 g, 183.9 mmol) in water (90 mL). The mixture was heated at reflux for 2 h., cooled, concentrated and acidified with hydrochloric acid (2M, 92.5 mL, 185 mmol). The dark precipitate was filtered, washed with water, and dried under vacuum. The solid was ground to a powder with a mortar and pestle, triturated with methanol (500 mL) then filtered again to yield 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoic acid as a muddy solid. This material was washed with ether, air dried overnight, and ground to a fine powder with mortar and pestle. On the basis of mass recovery (34.49 g) the yield was assumed to be quantitative. 1H NMR (300 MHz, d6-DMSO) δ 9.47 (1H, s); 8.53 (1H, d, J=5.2 Hz); 8.24 (2H, d, J=8.5 Hz); 8.08 (2H, d, J=8.8 Hz), 7.66 (2H, d, J=9.1 Hz); 7.37 (1H, d, J=5.2 Hz); 6.93 (2H, d, J=9.1 Hz); 3.73 (4H, m); 3.04 (4H, m). LC-ESI-MS (method C): rt 7.3 min.; m/z 377.1 [M+H]+.
To a suspension of 4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzoic acid (theoretically 32.59 g, 86.6 mmol) in DMF (400 mL) was added triethylamine (72.4 mL, 519.6 mmol, 6 eq.) The mixture was sonicated to ensure dissolution. Aminoacetonitrile hydrochloride (16.02 g, 173.2 mmol) was added followed by N-hydroxybenzotriazole (anhydrous, 14.04 g, 103.8 mmol) and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (19.92 g, 103.8 mmol). The suspension was stirred vigorously overnight. The solvent was evaporated under reduced pressure, the residue was diluted with 5% sodium bicarbonate (400 mL) and water (300 mL), giving a yellow solid, which was broken up and filtered. The solids were washed several times with 100 mL portions of water, triturated with hot methanol/dichloromethane (500 mL, 1:1), concentrated to a volume of approximately 300 mL), cooled and filtered. The solids were washed with cold methanol (3×100 mL), ether (200 mL) and hexane (200 mL) prior to drying to afford Compound 3 (31.69 g, 88%). M.p. 238-243° C. Microanalysis: Found C, 66.52; H, 5.41; N, 20.21. C23H26N6O10S2 requires C, 66.65; H, 5.35; N, 20.28%. 13C NMR (75.5 MHz, d6-DMSO) δ 166.04, 162.34, 160.26, 159.14, 146.14, 139.87, 134.44, 132.73, 127.80, 126.84, 120.29, 117.49, 115.50, 107.51, 66.06, 49.16, 27.68.
To a solution of 2,4-dichloro-5-methylpyrimidine (244 mg, 1.5 mmol) and methyl 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (210 mg, 1.0 mmol) in toluene (3 mL) were added n-propanol (1 mL), aqueous sodium bicarbonate (2 M, 1.5 μL) and tetrakis(triphenylphosphine)palladium[0] (116 mg, 0.1 mmol). The reaction was heated at 110° C. for 40 h, then partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice further with ethyl acetate and the combined organic fractions were washed with water, brine then dried (sodium sulfate), filtered and concentrated. Silica gel chromatography using 30-60% ethyl acetate/petroleum spirit as eluent provided methyl 4-(2-chloro-5-methylpyrimidin-4-yl)-2-methoxybenzoate as a cream solid (165 mg, 56%); LC-ESI-MS (method B): rt 6.2 min.; m/z 293.3/295.3 [M+H]+.
To a solution of methyl 4-(2-chloro-5-methylpyrimidin-4-yl)-2-methoxybenzoate (165 mg, 0.56 mmol) in 1,4-dioxane (5 mL) was added 4-morpholinoaniline (96 mg, 0.54 mmol) and p-toluenesulfonic acid monohydrate (97 mg, 0.51 mmol). The reaction was heated at reflux for 40 h, cooled to room temperature and partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice more with ethyl acetate and the combined organic fractions were washed twice with 5% aqueous citric acid, water, brine then dried (sodium sulfate) filtered and concentrated to afford the crude product. Trituration with methanol provided methyl 4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoate as a yellow solid (77 mg, 32%); 1H NMR (300 MHz, d6-DMSO) δ 9.36 (s, 1H), 8.38 (s, 1H), 7.76 (d, J=8.1 Hz, 1H), 7.62 (d, J=9.0 Hz, 2H), 7.38 (s, 1H), 7.27 (d, J=8.1 Hz, 1H), 6.88 (d, J=9.0 Hz, 2H), 3.89 (s, 3H), 3.82 (s, 3H), 3.72 (m, 4H), 3.01 (m, 4H), 2.12 (s, 3H); LC-ESI-MS (method B): rt 6.7 min.; m/z 435.3 [M+H]+.
To a solution of methyl 4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoate (70 mg, 0.16 mmol) in 1,4-dioxane (5 mL) was added aqueous sodium hydroxide (5 M, 5 mL). The reaction was heated at reflux overnight then cooled to room temperature. The yellow solid which precipitated was collected by filtration and washed with water to afford the sodium salt of Compound 40 in quantitative yield.
The sodium salt of 4-(2-(4-morpholinophenylamino)-5-methylpyrimidin-4-yl)-2-methoxybenzoic acid (Compound 40) (0.16 mmol), was acidified by suspending in ethyl acetate and partitioning against 5% aqueous citric acid. Further extraction with ethyl acetate followed by evaporation of the solvent then furnished the free acid which was suspended in dichloromethane (3 mL). To this solution was added triethylamine (111 μL, 0.8 mmol), 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (58 mg, 0.3 mmol), aminoacetonitrile hydrochloride (61 mg, 0.4 mmol) and a catalytic amount of N,N-dimethyl aminopyridine. N,N-Dimethyl formamide (2 mL) was added to aid solubility and the reaction was stirred for 64 h. The reaction was incomplete by TLC analysis so 0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (76 mg, 0.2 mmol) was added and the reaction stirred for a further 24 h before being partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice further with dichloromethane and the combined organics washed with water, brine then dried (sodium sulfate) filtered and concentrated to afford the crude product. Silica gel chromatography using 0-3% methanol/ethyl acetate as the eluent afforded, as a green/yellow solid, Compound 47 (13.2 mg, 18%).
To a suspension of 4-carboxyphenylboronic acid (5.0 g, 30 mmol) in DMF (5 mL) and dichloromethane (200 mL) at 0° C. was added oxalylchloride (5.9 mL, 66 mmol) dropwise. When gas evolution slowed, the ice bath was removed and the reaction allowed to warm to room temperature over 30 min. The reaction was then heated at 40° C. for three hours by which time all solids had dissolved. The dichloromethane was removed by distillation and the DMF solution cooled to 0° C. A solution of aminoacetonitrile hydrochloride (3.05 g, 33 mmol) in DMF (80 mL) and DIPEA (13 mL, 75 mmol) was then added dropwise. After the addition was complete the ice bath was removed and the solution allowed to stir at room temperature for 16 h. Most of the DMF was then removed in vacuo and the reaction was partitioned between ethyl acetate and 2 M aqueous hydrochloric acid. The aqueous layer was extracted twice further with ethyl acetate and the combined organic fractions dried (Na2SO4) filtered and concentrated under reduced pressure to afford 4-(cyanomethylcarbamoyl)phenylboronic acid as a waxy pale yellow solid (5.34 g, 87%). 1H NMR (300 MHz, d6-DMSO): 9.18 (br. t, J=5.1 Hz, 1H), 7.8-7.9 (m, 4H), 4.31 (d, J=5.4 Hz, 2H); LC-ESI-MS (method B): rt 0.9 min.; m/z 203.3 [M−H]−.
To a solution of 2,4-dichloropyrimidine (3.2 g, 0.22 mmol) and 4-(cyanomethylcarbamoyl)phenylboronic acid (3.0 g, 15 mmol) in toluene (146 mL) were added n-propanol (44 mL), aqueous sodium bicarbonate (2M, 22 mL) and tetrakis(triphenylphosphine) palladium[0] (850 mg, 0.7 mmol). The reaction was heated at 90° C. for 24 h, then partitioned between ethyl acetate and water. The aqueous layer was extracted twice further with ethyl acetate and the combined organic fractions washed with brine, dried (Na2SO4) filtered and concentrated. Silica gel chromatography using 30-70% ethyl acetate/petroleum spirit as eluent provided 4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide as a pale yellow waxy solid (1.35 g, 33%). 1H NMR (300 MHz, d6-DMSO) δ 9.40 (t, J=5.4 Hz, 1H), 8.88 (d, J=5.2 Hz, 1H), 8.32 (d, J=8.7 Hz, 2H), 8.23 (d, J=5.1 Hz, 1H), 8.05 (d, J=8.7 Hz, 2H), 4.36 (d, J=5.4 Hz, 2H); LC-ESI-MS (method B): rt 5.3 min.; m/z 273.2/275.2 [M+H]+.
A Schlenck flask was dried with a heat gun under vacuum for two minutes and then backfilled at room temperature with nitrogen. Tris(dibenzylideneacetone)dipalladium (9 mg, 0.01 mmol), (2-biphenylyl)di-tert-butylphosphine (5.7 mg, 0.02 mmol), potassium phosphate (56 mg, 0.27 mmol), 4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide (52 mg, 0.19 mmol) and 4-[(1,1-dioxidothiomorpholin-4-yl)methyl]aniline (40 mg, 0.17 mmol) were added and mixed together in the flask under a constant flow of nitrogen. The flask was sealed, evacuated under high vacuum and then backfilled with nitrogen. The operation was repeated twice. 1,2-dimethoxyethane (1.9 mL) was added through the rubber septum. The flask was sealed and vigorous stirring was initiated. The mixture was then frozen with liquid nitrogen, degassed under high vacuum and then backfilled with nitrogen (the operation was repeated twice). The sealed flask was then heated to 100° C. overnight. A small amount of tris(dibenzylideneacetone) dipalladium and (2-biphenylyl)di-tert-butylphosphine was added, the mixture was frozen, degassed under high vacuum and backfilled with nitrogen before being heated at 100° C. for a further 16 h. Ethyl acetate was added and the mixture filtered through a sintered funnel. The filtrate was then concentrated and ethyl acetate added. The resulting mixture was then washed with a solution of citric acid (2%) and a saturated solution of sodium chloride. The organic layer was dried (sodium sulfate), filtered and evaporated to give the crude product which was purified by column chromatography using petroleum spirit/ethyl acetate (1/4) to give a residue which was triturated with methanol to give Compound 90 (5.5 mg, 7%).
A round bottomed flask was charged with 4-methanesulfonylaminophenylboronic acid (4.30 g, 20 mmol) and 2,4-dichloropyrimidine (5.97 g, 40 mmol, 2 eq.), toluene (75 mL), n-propanol (25 mL) and aqueous sodium carbonate solution (2M, 18 mL, 1.8 eq.). The reaction mixture was evacuated and backfilled with nitrogen three times before adding tetrakis(triphenylphosphine) palladium (0) catalyst (1.02 g, 4.4 mol %). The reaction mixture was again evacuated and backfilled with nitrogen three times before being heated at 100° C. under a nitrogen atmosphere for 66 hours. The reaction mixture was cooled and stirred at room temperature for several hours during which time the product precipitated from the reaction mixture. The fine yellow solid (3.45 g, 61% yield) was collected by vacuum filtration, washed with methanol and dried under high vacuum. 1H NMR and LC MS data confirmed this to be the desired N-(4-(2-chloropyrimidin-4-yl)phenyl)methanesulfonamide. 1H NMR (300 MHz, d6-DMSO) δ 10.26 (1H, brs); 8.75 (1H, d, J=5.5 Hz); 8.17 (2H, d, J=9.1 Hz); 8.05 (1H, d, J=5.5 Hz); 7.35 (2H, d, J=8.7 Hz); 3.10 (3H, s). LC-ESI-MS (method B): rt 5.5 min.; m/z 284.2/286.1 [M+H]+.
N-(4-(2-chloropyrimidin-4-yl)phenyl)methanesulfonamide (750 mg 2.64 mmol) and potassium carbonate (730 mg, 2 eq.) were placed in a round bottomed flask and suspended in acetone (50 mL). The mixture was stirred for several minutes before adding bromoacetonitrile (368 μL, 2 eq.). The reaction mixture was stirred at room temperature for 48 h. The crude reaction mixture was concentrated in vacuo and the residue taken up in ethyl acetate (200 mL) and washed with water (2×100 mL), brine (100 mL) and then dried (sodium sulfate). The organic phase was concentrated in vacuo to give N-(4-(2-chloropyrimidin-4-yl)phenyl)-N-(cyanomethyl)methanesulfonamide (762 mg, 89% yield) as a fawn solid. 1H NMR (300 MHz, d6-DMSO) δ 8.86 (1H, d, J=5.0 Hz); 8.28 (2H, d, J=8.7 Hz); 8.18 (1H, d, J=5.5 Hz); 7.66 (2H, d, J=8.7 Hz); 4.97 (2H, s); 3.22 (3H, s). LC-ESI-MS (method B): rt 5.9 min.; m/z 323.2/325.2 [M+H]+.
N-(4-(2-chloropyrimidin-4-yl)phenyl)-N-(cyanomethyl)methanesulfonamide (171 mg, 0.53 mmol), 5-amino-2-morpholinobenzoic acid (142 mg, 1.2 eq.) and p-toluene sulfonic acid monohydrate (98 mg, 0.98 eq.) were suspended in 1,4-dioxane (8 mL) and heated at 100° C. overnight. The reaction mixture was cooled to room temperature, concentrated in vacuo. The residue was taken up in ethyl acetate (80 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was then dried and concentrated in vacuo. The residue was repeatedly triturated with methanol (5 mL then 3 mL) to afford, as a cream solid, 5-(4-(4-(N-(cyanomethyl)methylsulfonamido)phenyl)pyrimidin-2-ylamino)-2-morpholinobenzoic acid (101 mg, 37%). 1H NMR (300 MHz, d6-DMSO) δ 17.23 (1H, s) CO2H; 9.99 (1H, s); 8.74 (1H, d, J=2.7 Hz); 8.62 (1H, d, J=5.0 Hz); 8.33 (2H, d, J=8.7 Hz); 8.01 (1H, dd, J=2.8, J=8.7 Hz); 7.69 (1H, d, J=9.1 Hz); 7.63 (2H, d, J=8.7 Hz); 7.52 (1H, d, J=9.1 Hz); 4.97 (2H, s); 3.81 (4H, m); 3.21 (3H, s); 3.06 (4H, m). LC-ESI-MS (method C): rt 5.4 min.; m/z 509.3 [M+H]+.
5-(4-(4-(N-(Cyanomethyl)methylsulfonamido)phenyl)pyrimidin-2-ylamino)-2-morpholinobenzoic acid (50 mg, 0.098 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (41 mg, 1.1 eq.) were dissolved in anhydrous N,N-dimethylformamide (4 mL) and sonicated for 5 minutes. Triethylamine (41 mL, 3 eq.) and N,N-dimethylethylenediamine (21 mL, 2 eq.) were added and the mixture stirred overnight at room temperature. The reaction mixture was then diluted with ethyl acetate (50 mL) and washed with bicarbonate solution (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried (sodium sulfate) and concentrated in vacuo to afford, as a yellow solid, Compound 73 (48 mg, 86% yield).
To a solution of 5-bromo-2,4-dichloropyrimidine (300 mg, 1.3 mmol) in dichloromethane (3 mL) kept at −5° C. was added cold 57% aqueous hydroiodic acid (5 mL). The resulting solution was stirred at −5° C. for 2 hours. Solid sodium carbonate was added in small portions until the solution was pH 7 and the mixture was decolourised by adding 5% aqueous sodium metabisulphite. Water was added until the entire solid dissolved and the organic phase was separated. The aqueous phase was extracted twice with dichloromethane then the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give crude 5-bromo-2,4-diiodopyrimidine as a white solid (410 mg). This material was used for the next step without further purification. LC-ESI-MS (method B): rt 6.8 min.; m/z 410.9/412.9 [M+H]+.
To a mixture of 4-(cyanomethylcarbamoyl)phenylboronic acid (see example 3) (185 mg, 0.9 mmol) and 5-bromo-2,4-diiodopyrimidine (410 mg, 1.0 mmol) in 1,4-dioxane (10 mL), was added 2M aqueous potassium carbonate (100 μL). The resulting mixture was stirred under nitrogen for 5 minutes then tetrakis(triphenylphophine)palladium(0) (52 mg, 0.045 mmol) was added under a nitrogen atmosphere. The mixture was heated at 80° C. overnight. The cooled reaction mixture was diluted with water and extracted twice with ethyl acetate. The combined organic extracts were washed with water then brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product as a brown solid. The crude material was purified by flash chromatography, eluting with 50% ethyl acetate/petroleum spirit to give 4-(5-bromo-2-iodopyrimidin-4-yl)-N-(cyanomethyl)benzamide (200 mg, 35% over 2 steps). LC-ESI-MS (method B): rt 6.2 min.; m/z 443.0/445.0 [M+H]+.
To a round bottom flask containing 4-(5-bromo-2-iodopyrimidin-4-yl)-N-(cyanomethyl)benzamide (45 mg, 0.1 mmol) and 4-morpholinoaniline (27 mg, 0.15 mmol) in 1,4-dioxane (3 mL), was added diisopropylamine (26 mg, 0.2 mmol). The flask was equipped with a reflux condenser and the reaction mixture was heated at reflux overnight. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water then brine, dried over anhydrous sodium sulfate and concentrated to give the crude product as a brown solid. The crude material was purified by flash chromatography, eluted with 50% ethyl acetate/petroleum spirit then 80% ethyl acetate/petroleum spirit to give, as a yellow solid, Compound 65 (12 mg, 24%).
Compound 3 (10.0 g) was suspended in methanol (1 L). Concentrated sulfuric acid (10.52 g, 90% w/w) was added dropwise to the stirring solution. A clear brown solution resulted and a solid lump formed. The solution was filtered quickly then allowed to continue stirring for 3 h (a second precipitate appeared within minutes). After this time the pale yellow precipitate was collected by filtration, washed with methanol (10 mL) then dried under vacuum overnight to afford 4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-ium hydrogensulfate, as a pale yellow solid (10.20 g, 69%). m.p. 205° C. Microanalysis: Found C, 45.18; H, 4.36; N, 13.84; S, 10.24. C23H26N6O10S2 requires C, 45.24; H, 4.29; N, 13.76; S, 10.50%. 1H NMR (300 MHz, d6-DMSO) δ 9.85 (br. s, 1H), 9.34 (t, J=5.4 Hz, 1H), 8.59 (d, J=5.2 Hz, 1H), 8.27 (d, J=8.5 Hz, 2H), 8.03 (d, J=8.5 Hz, 2H), 7.83 (d, J=8.4 Hz, 2H), 7.50 (d, J=5.2 Hz, 1H), 7.34 (br. s, 2H), 4.36 (d, J=5.4 Hz, 2H), 3.89 (br. s, 4H), 3.37 (br. s, 4H); 13C NMR (75.5 MHz, d6-DMSO) δ 166.07, 163.36, 159.20, 158.48, 140.19, 139.34, 136.45, 134.89, 128.00, 127.22, 121.13, 119.89, 117.59, 109.05, 64.02, 54.04, 27.82. LC-ESI-MS (method D): rt 10.0 min.; m/z 415.1 [M+H]+.
Compound 3 (0.25 g) was suspended in methanol (25 ml). Methane sulfonic acid (0.255 g) was added dropwise to the stirring solution and a clear brown solution resulted. The solution was allowed to stir for 3 h, after which the volume was reduced to 9 ml. The resultant precipitate was collected and dried under vacuum for 8 h to afford 4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-ium methanesulfonate as a pale yellow solid (0.22 g). m.p. 208° C. 1H NMR (300 MHz, d6-DMSO) δ 9.83 (br. s, 1H), 9.35 (t, J=5.3 Hz, 1H), 8.59 (d, J=5.1 Hz, 1H), 8.28 (d, J=8.5 Hz, 2H), 8.04 (d, J=8.5 Hz, 2H), 7.83 (d, J=9.0 Hz, 2H), 7.50 (d, J=5.5 Hz, 1H), 7.31 (d, J=9.0 Hz, 2H), 4.36 (d, J=5.5 Hz, 2H), 3.88 (m, 4H), 3.35 (br. s, 4H), 2.36 (s, 6H); LC-ESI-MS (method D): rt 10.2 min.; m/z 415.3 [M+H]+.
Compound 3 (0.50 g) was suspended in methanol (45 ml). A freshly prepared solution of hydrochloric acid in methanol (2.6 ml, HCl conc. 40 mg/ml) was added dropwise to the stirring solution and a clear brown solution resulted. The solution was allowed to stir for 2 h, then the resultant precipitate was collected, washed with methanol (5 ml) and dried under vacuum for 8 h to afford 4-(4-(4-(4-(cyanomethylcarbamoyl)phenyl)pyrimidin-1-ium-2-ylamino)phenyl)morpholin-4-ium chloride a pale yellow solid (0.30 g). m.p. 210° C. 1H NMR (300 MHz, d6-DMSO)1H NMR (300 MHz, DMSO) δ 9.92 (br. s, 1H), 9.42 (t, J=5.3, 1H), 8.62 (d, J=4.8, 1H), 8.29 (d, J=8.1, 2H), 8.06 (d, J=8.1, 2H), 7.89 (d, J=9.0, 2H), 7.53 (br. s, 3H), 4.36 (d, J=5.4, 2H), 3.82 (br. s, 4H), 3.43 (br. s, 4H). LC-ESI-MS (method D): rt 10.3 min.; m/z 415.3 [M+H]+.
A 50 mL two necked round bottom flask was fitted with a magnetic stirrer bar and a dropping funnel. A suspension of NaBH4 in tetrahydrofuran (100 mg, 2.4 mmol/10 mL) was added, followed by 5-amino-2-morpholinobenzenecarboxylic acid (222 mg, 1.0 mmol) in one portion. A reflux condenser was fitted and the reaction mixture was cooled to 0° C. under nitrogen atmosphere. A solution of iodine in tetrahydrofuran (250 mg, 1.0 mmol/15 mL) was added dropwise to the reaction mixture. After iodine addition was completed and gas evolution had ceased, the reaction mixture was heated at reflux for 6 hours and left stirring at room temperature overnight. Methanol was added slowly until the mixture became clear. The resulting solution stirred at room temperature for 30 minutes then solvent was removed under reduced pressure. The residue was dissolved in 20% KOH (30 mL), stirred for 4 hours and extracted with dichloromethane (3×30 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4 and concentrated to give 5-amino-2-morpholinobenzyl alcohol as an off-white solid (150 mg, 72% yield). 1H NMR (300 MHz, CDCl3) δ 7.05 (d, J=8.7 Hz, 1H), 6.59 (dd, J=8.4, 2.7 Hz, 1H), 6.49 (d, J=2.7 Hz, 1H), 5.51 (br s, 1H), 4.71 (s, 2H), 3.84 (t, J=5.1 Hz, 4H), 3.60 (br s, 2H), 2.91 (t, J=5.1 Hz, 4H). LC-ESI-MS (method B): rt 2.31 min.; m/z 209.2 [M+H]+.
To a suspension of NaH in cold tetrahydrofuran (80 mg/20 mL), 5-amino-2-morpholinobenzyl alcohol (400 mg, 2 mmol) was added. The mixture was stirred for 15 minutes then allyl chloride (150 mg, 2 mmol) and tetrabutylammonium iodide (37 mg, 5 mol %) were added. The resulting mixture was stirred at room temperature for 2 hours then at 60° C. overnight. After cooling to room temperature, water was added (200 μL) and the mixture stirred for 10 minutes then diluted with ethyl acetate. The organic phase was washed sequentially with 10% aqueous ammonium chloride and brine, dried over anhydrous Na2SO4, filtered and concentrated to give a yellow solid. The crude product was purified with 50% ethyl acetate in petroleum spirit to obtain 3-((allyloxy)methyl)-4-morpholinobenzenamine as a light orange oil (250 mg, 50% yield). 1H NMR (300 MHz, CDCl3) δ 6.95 (d, J=8.3 Hz, 1H), 6.82 (d, J=2.7 Hz, 1H), 6.61 (dd, J=8.2, 2.7 Hz, 1H), 6.10-5.90 (m, 1H), 5.34-5.28 (m, 1H), 5.23 (dd, J=10.5, 1.9 Hz, 1H), 4.57 (s, 2H), 4.07-4.05 (m, 2H), 3.80 (t, J=4.8 Hz, 4H), 3.55 (br s, 2H), 2.83 (t, J=4.6 Hz, 4H). LC-ESI-MS (method B) rt 5.91 min.; m/z 249.3 [M+H]+.
3-((allyloxy)methyl)-4-morpholinobenzenamine was converted to Compound 79 by reaction with 4-(2-chloropyrimidin-4-yl)-N-(cyanomethyl)benzamide in the presence of p-toluene sulfonic acid using methods analogous to those described for the synthesis of Compound 3 and Compound 47.
Compound Analysis
1H and 13C NMR data were acquired on a Brucker AV-300 AVANCE NMR spectrometer.
LC-EI-MS and EI-MS
General Parameters:
LC-EI-MS and EI-MS data were acquired on a Waters 2795 Alliance HPLC coupled to a Waters 2996 Photodiode Array Detector and Integrity TMD Electron Impact Mass Spectrometer operating under control of Waters Millenium32 software version 4.0 with the settings outlined below.
Mass Spectrometer Parameters:
Helium flow of approximately 0.36 L/min.; acquisition mode set to scan; sampling rate of 1 spectra/sec; source temperature 200° C.; nebuliser temperature 80° C.; expansion region temperature 75° C.; mass range m/z 100-550, m/z 100-650 or m/z 100-700 as required.
HPLC Parameters
LC-MS parameters were as described for each of the methods outlined below. EI-MS samples were injected and analysed with no column present, with a solvent flow rate of 0.25 mL/min.
Method A1 (LC-EI-MS)
Solvent Gradient:
Flow rate: 0.25 mL/min
Column: one of
Method A2 (LC-EI-MS)
Solvent Gradient:
Flow rate: 0.25 mL/min
Column: one of
LC-ESI-MS
General Parameters:
LC-ESI-MS data was acquired on a Waters 2695Xe HPLC coupled to a Waters 2996 Photodiode Array Detector and Waters ZQ Mass Spectrometer operating under electrospray ionization conditions with Masslynx software version 4.1 with the settings outlined below.
Mass Spectrometer Parameters:
Mass range: m/z 100-650
Scan time: 0.5
Inter scan delay: 0.1
Desolvation gas: 500 L/h N2
Cone Gas: 100 L/h N2
Desolvation Temperature: 400° C.
Source Temperature: 120° C.
Cone Voltage: +30 V for ESI positive mode, or
HPLC Parameters:
Were one of the following sets of conditions outlined below.
Method B
Solvent Gradient:
Flow rate: 0.25 mL/min.
Column: XTerra MS C18, 2.1×50 mm, 3.5 micron
Method C
Solvent Gradient:
Flow rate: 0.25 mL/min.
Column: XTerra MS C18, 2.1×50 mm, 3.5 micron
Method D
Solvent Gradient:
Flow rate: 0.25 mL/min.
Column: XTerra MS C18, 3.0×100 mm, 3.5 micron
Compound Dilution
For screening purposes, compounds (in 100% DMSO) were warmed at 37° C. for at least 20 minutes before use. A 20 μm stock was initially made in assay buffer, where the final concentration of DMSO was 0.3%. The stocks were then diluted in 384 well Optiplates (Packard) where the final concentration of the compound was 5 μM.
JAK Tyrosine Kinase Domain Production
JAK kinase domains were produced using the following procedures:
JAK2
The kinase domain of human JAK2 was amplified from U937 mRNA using the polymerase chain reaction with the following primers:
The JAK2 PCR products were cloned into the pDest20 destination vector (Gibco). The JAK2 plasmid was then transformed into competent DH10Bac cells (Gibco), and the recombinant baculovirus was prepared via Sf9 insect cell transfection.
JAK3
The kinase domain of human JAK3 was amplified from U937 mRNA using the polymerase chain reaction with the following primers:
The JAK3 PCR products were cloned into the pDest20 destination expression vector (Gibco). The JAK3 plasmid was then transformed into competent DH10Bac cells (Gibco), and the recombinant baculovirus was prepared via Sf9 insect cell transfection.
Large Scale Production of Kinase Domains
Baculovirus preparations from each of the JAK family members were infected into one litre of Sf9 (Spodoptera frugiperda) cells (Invitrogen) grown in SF900II serum free medium (Invitrogen) to a cell density of approximately 2×106 cells/ml. Cells were infected with virus at a cell culture to virus stock ratio of 20:1. Cells were harvested and lysed 48 hours post infection. The GST-tagged JAK kinase domains were purified by affinity chromatography on a GSH agarose column (Scientifix).
Assay Protocols
Kinase assays were performed in 384 well Optiplates (Packard) using an Alphascreen Protein Tyrosine KinaseP100 detection kit The compounds were pre-incubated with affinity purified PTK domain in the presence of phosphotyrosine assay buffer (10 mM HEPES, pH 7.5, 100 mM MgCl2, 25 mM NaCl, 200 mM sodium vanadate and 0.1% Tween® 20) for 20 minutes. The compounds were then incubated with substrate in the presence of either 80 or 625 um ATP for 60 or 90 minutes. The substrate used was either substrate-1 with the sequence biotin-EGPWLEEEEEAYGWMDF-NH2 [SEQ ID NO:13] (final concentration 111 μM) or substrate-2 substrate with the sequence biotin-EQEDEPEGDYFEWLEPE (final concentration 133 μM). Alphascreen phosphotyrosine acceptor beads followed by streptavidin donor beads at a concentration of 1/100 in stop buffer were added to each well under subdued light and incubated for 2-3 hours. The Alphascreen plates were read on a Packard Fusion Alpha instrument
The enzyme assay results and structural data for selected compounds is given below in Table 2, where +++ is <100 nM, ++ is <500 nM and + is <1 μM
Compound Dilution
For screening purposes, compounds were diluted in 96 well plates at a concentration of 20 μM. Plates were warmed at 37° C. for 30 minutes before the assay was performed.
Establishment of the TEL:JAK2 Cell Line
The coding region encompassing nucleotides 1-487 of TEL was amplified by PCR using the oligonucleotides 5TEL (5′-GGA GGA TCC TGA TCT CTC TCG CTG TGA GAC-3′) [SEQ ID NO 14] and 3TEL (5′-AGGC GTC GAC TTC TTC TTC ATG GTT CTG-3′) [SEQ ID NO 15] and U937 mRNA as a template. A BamHI restriction site was incorporated into the 5TEL primer, and a Sal I restriction site was incorporated into the 3TEL primer. The regions encompassing the kinase domain of JAK2 (nucleotides 2994-3914; JAK2F 5′-ACGC GTC GAC GGT GCC TTT GAA GAC CGG GAT-3′ [SEQ ID NO 16]; JAK2R 5′-ATA GTT TAG CGG CCG CTC AGA ATG AAG GTC ATT T-3′) [SEQ ID NO 17] and JAK3 (nucleotides 2520-3469; JAK3F 5′-GAA GTC GAC TAT GCC TGC CAA GAC CCC ACG ATC TT-3′) [SEQ ID NO 18] were generated by PCR using Taq DNA polymerase (Gibco/BRL) and U937 mRNA as a template. A Sal I restriction site was incorporated into the forward primer of JAK2 and JAK3, a Not I site was incorporated into the JAK2 reverse primer and a Xba I site was added to the reverse primer of JAK3.
A TEL/Jak2 fusion was generated by digestion of the TELPCR product with BamH I/Sal I restriction enzymes, digestion of the JAK2 PCR product with Sal I/Not I restriction enzymes, followed by ligation and subcloning of the ligation product into the mammalian expression Vector pTRE 2 (Clontech), which was prepared by digestion with BamH I-Not I restriction enzymes, to give the TEL/Jak2 fusion plasmid pTELJAK2.
The TEL/Jak3 fusion was prepared by ligation of the JAK3 Sal I/Not I cleaved kinase domain PCR product with the BamH I/Sal I restriction digested TEL product, followed by ligation of the ligation product into the BamH I/Not I digested pTRE2, to give the TEL/Jak3 fusion plasmid pTELJAK3.
The growth factor dependent myelomonocytic cell line BaF3 bearing the pTET-off plasmid (Clontech) was transfected with either pTELJAK2 or pTELJAK3, and the transfected cells were selected for growth-factor independent cell growth. The BaF3 wild-type cells were cultured in DMEM containing 10% FCS, 10% WEHI 3B conditioned medium. The BaF3 TELJAK cells (BafT_J2 or BafT_J2) were cultured in DMEM 10% Tet-System Approved FBS (without WEHI 3B conditioned medium).
Cellular assays were performed as follows:
Cell suspensions were prepared by harvesting cells from culture (the cells used in this test were in late log phase growth with high viability.) Cells were diluted in the appropriate growth medium, as described above, to 1.1× final concentration (from 50,000 cell/mL to 200,000 cell/mL, depending on cell line).
Compounds to be tested were added (10 μL, 10× final concentration) to a flat bottomed 96-well plate. The cellular suspension (90 μL per well) was then added, and the plate incubated for 48-72 hr at 37° C., 5% CO2. Alamar Blue 10 μL per well was added and the plates returned to the incubator for a further 4-6 hours. The plates were then read at 544 nm.
Results
Result are given in table 2 where +++ is <1 μM, ++ is <5 μM and + is <20 μM
Multiparameter Intracellular Flow Cytometric Analysis of STAT 5 Phosphorylation.
The human erythroleukaemic cell line, HEL 92.1.7 (ATCC, TIB-180), was grown in RPMI 1640 containing 10% FCS supplemented with 1 mM sodium pyruvate. For phosphor-STAT 5 determination, HEL cells were grown in RPMI 1640+1% FCS for 18 hours at 37° C. and 2×105 cells per assay point were exposed to DMSO/test compounds for 2 hours at 37° C. The cells were centrifuged at 1300 rpm for 3 minutes and fixed in paraformaldehyde (2% final concentration) for 15 minutes at 37° C. After centrifugation, cells were permeabilized in 90% methanol at 4° C. for 30 minutes. Following three washes in PBS-2% FCS, the staining was performed as follows using BD PharMingen phycoerythrin-conjugated mouse immunoglobulin isotype control (Cat. No. 551436 and phycoerythrin-conjugated mouse IgG1 antibody to STAT 5 (Y694) (Cat. No. 612567).
Staining proceeded for 1 hour at room temperature in the dark, followed by 3 washes in PBS-2% FCS. The cells were next resuspended in 800 μL PBS-FCS for FACS analysis. Flow cytometry was performed using a Beckman Cell Lab Quanta SC System with 3 colour and side scatter capabilities. Data analysis was performed with CXP analysis software (version 2.2). The median fluorescence intensity (MFI) was used to determine fold change upon treatment of cells with specific inhibitor compounds, calculated as the MFIstimulated/MFIunstimulated ratio for the phosphospecific antibody fluorescence channel (FL2).
The results shown in
Experiment 1
Methodology
The murine pro-B cell line BaF3 was routinely maintained in RPMI 1640 media containing 10% FCS. On the day of the experiment, cells were washed twice in PBS, and resuspended in RPMI 1640 media containing 0.1% FCS. After 2 hours of serum deprivation, cells were treated with the desired concentration of Compound 3, Control Compound, or vehicle alone (DMSO) for a further 2 hours. Mouse IL-3 was then added to cells at a final concentration of 5 ng/ml for 15 minutes. Cells were then placed on ice and washed twice in ice-cold PBS. Washed cell pellets were snap-frozen in liquid nitrogen and stored at −80° C.
Cell pellets were lysed on ice in RIPA buffer, and lysates clarified by centrifugation (20,000×g, 4° C., 5 min). The protein concentration of lysates was determined by the Bradford method, and equal amounts of protein (60 μg/lane) were separated by SDS-PAGE. Protein was then transferred to PVDF, and Western blotting performed using an antibody that specifically recognizes STAT5 phosphorylated at tyrosine 694. The membrane was then stripped and reprobed with an antibody that recognizes total STAT5 protein.
The results shown in
Experiment 2
Methodology
The human erythroleukaemic cell line HEL 92.1.7 was routinely maintained in RPMI 1640 media containing 10% FCS. The day before the experiment, cells were washed twice in PBS, resuspended in RPMI 1640 media containing 1% FCS, and cultured overnight.
The following day, cells were treated with the desired concentration of Compound 3, Control Compound, or vehicle alone (DMSO) for 2 hours. Cells were then placed on ice and washed twice in ice-cold PBS. Washed cell pellets were snap-frozen in liquid nitrogen and stored at −80° C.
Cell pellets were lysed on ice in RIPA buffer, and lysates clarified by centrifugation (20,000×g, 4° C., 5 min). The protein concentration of lysates was determined by the Bradford method, and equal amounts of protein (60 μg/lane) were separated by SDS-PAGE. Protein was then transferred to PVDF, and Western blotting performed using an antibody that specifically recognizes STAT5 phosphorylated at tyrosine 694. The membrane was then stripped and reprobed with an antibody that recognizes total STAT5 protein.
The results shown in
The effect of Compound 3 on growth hormone-stimulated insulin-like growth factor-1 concentrations in mouse plasma.
Circulating IGF-1 concentrations (mean±s.e.m.) in female C3/H mice (n=6/group) after administration of compound 3 (50 mg/kg), or vehicle only (Control, +GH), by oral gavage 8 h and 30 min prior to subcutaneous administration of growth hormone (+GH, 30 μg/mouse) or saline (Control) at time 0. Blood samples were collected 6 h post −GH administration, and plasma IGF-1 concentrations measured using an ELISA for mouse IGF-1 (R & D Systems). Different superscripts denote significant differences (p<0.05) between groups detected by one-way ANOVA and Bonferroni's test post-hoc.
The results shown in
Efficacy of orally administered Compound 3 in a subcutaneous tumour model of Ba/F3 TelJAK2 cells in nude mice.
Balb/Cnu/nu mice were inoculated subcutaneously with mouse Ba/F3 TelJAK2 cells (2.5×106/mouse), and dosing b.i.d. by oral gavage with compound 3 (20 mg/kg, 10 mg/kg, or 5 mg/kg), or vehicle only (5% N-methylpyrrolidone, 0.1 M Captisol®), or Taxol® (5 mg/kg i.v. 3× weekly, n=15 mice/group). Dosing commenced 11 days post-tumour cell inoculation, when tumours were palpable (mean tumour volume of 6 mm3). Tumour dimensions were measured twice weekly. By dosing day 14, the mean percentage T/C values were 39% for compound 3 at 20 mg/kg b.i.d., 25% at 10 mg/kg b.i.d., and 82% at 5 mg/kg/day. Comparison of tumour volumes after 14 days of dosing by t-test (Mann Whitney Rank Sums Tests) found smaller tumour volumes (p<0.05) in groups treated with compound 3 at 20 mg/kg b.i.d., and 10 mg/kg b.i.d., and Taxol, compared to the Vehicle Control Treated Group and the 5 mg/kg Compound 3 Treated Group, which were not different from each other. A more stringent statistical test (Kruskal Wallis One way ANOVA) followed by Dunn's multiple comparison against the Control Group post-hoc identified a significant difference (p<0.05) between the Vehicle Control Treated Group and either the Compound 3 Treated Group (either 10 or 20 mg/kg b.i.d.) or the Taxol treated Group. The results are shown in
The results show that compound 3 inhibits the JAK2 enzyme in vitro, as well as the in vitro growth of Baf3Tel Jak2 cells, which are dependent on constitutively active Jak2 for growth and survival. Baf3Tel JAK2 cells growing in vivo as a tumour, are also inhibited by compound 3 in a dose-dependent manner. In addition the results demonstrate that compound 3 inhibits growth hormone (and therefore JAK2 dependent)-driven IGF-1 synthesis and secretion from the mouse liver in vivo.
The compounds can also be tested in a murine model of JAK2V617F-positive myeloproliferative disease (MPD)
Establishment of JAK2V617F-Positive MPD
Bone marrow from male 5-Flurouracil-treated Balb/c mice could be infected with a JAK2-V617F-GFP retrovirus and retroorbitally injected into lethally irradiated female recipients. From day 21 on the mice could be monitored by daily inspection and twice weekly blood counts+FACS for GFP-positive cells. It would be expected that a rise in hematocrit could occur around day 28 and a rise of the white blood cell count around day 40.
Treatment with Compounds
Early intervention group: Treatment would start on day 21 with compound or carrier given per oral gavage (12 mice in each group). Mice could be monitored by daily inspection and twice weekly blood counts+FACS for GFP-positive cells. Animals would be sacrificed on day 60 8-12 h after the last drug dose. Moribund mice or mice with a white cell count over 200,000/nl or weight loss>20% could be sacrificed earlier.
Late intervention group: Groups of 3 mice could be sacrificed on day 29, 36, 43, 50 and 57 and bone marrow and spleen could be analyzed for reticulin fibrosis. Treatment could start with compound or carrier given per oral gavage as soon as fibrosis is documented in 3/3 mice. Mice could be monitored by daily inspection and twice weekly blood counts+FACS for GFP-positive cells. Animals could be sacrificed after 30 days of therapy 8-12 h after the last drug dose. Moribund mice or mice with a white cell count over 200,000/nl or weight loss>20% could be sacrificed earlier. Animals could be subjected to necropsy.
Analysis of Tissues and Survival
Liver and spleen weights could be determined. Tissue sections from bone marrow, liver and spleen could be analyzed by HE stain. Marrow and spleens could also be silver-stained to assess reticulin fibrosis. Spleen and marrow cells could be analyzed by FACS for GFP, lineage markers, JAK2 and STAT5 phosphorylation. Blood could be collected by heart puncture and plasma separated and frozen for drug concentration measurement. Survival between groups could be compared with the Kaplan-Meyer method.
Assessment of the Activity of JAK2 Inhibitors in Colony-Forming Assays of Human Hematopoietic Cells
Peripheral blood mononuclear cells from patients with MPD (predominantly myelofibrosis) with and without JAK2V617F mutation (N=10 for each) and 5 normal controls (commercial supplier) could be isolated by density gradient centrifugation (Ficoll). CD34+ cells can be selected using commercial kits to enrich for progenitor cells. CD34+ cells can be plated in triplicate in methylcellulose supplemented with fetal bovine serum and cytokines (+/− EPO). After incubation of the plates for 2 weeks erythroid and myeloid colony formation could be assessed under an inverted microscope.
Cancer
The effect of the compounds on tumor initiation, progression and metastasis can be evaluated in relevant in vivo animal efficacy models. Models could be human tumor xenografts models in immuno-deficient mice, from human tumor cell lines or preferably from primary or metastatic human tumors. Other models might be human tumor xenografts grown in orthotopic sites, models of disseminated disease and transgenic or labeled tumors models. Models could also include surgical resection of primary tumor and evaluation of metastatic disease.
Models could be selected to ensure that the molecular drug targeted is expressed. Examples of tumors displaying deregulation of the JAK/STAT pathway include prostate carcinoma, breast cancer, colon carcinoma, including leukemia, lymphoma, myeloma, ovarian tumors, melanoma, lung carcinoma, glioma, renal-cell tumors.
Efficacy can be measured in these models by various outcomes depending on tumor type (solid, leukemia or metastatic) and might include measure of tumor onset, tumor growth rate, tumor burden, tumor growth delay, tumor cell kill, incidence of metastasis, imaging of tumor and invasiveness/metastasis by various approaches including labeled cells or reagents, survival, angiogenesis, histopathology.
The in vivo animal efficacy models might also be used for determination of the additivity or synergy of the effect of the compounds in combination with other drugs,
Asthma is restricted to human species, but animal models are often used to investigate particular aspects of this human disease. Bronchial biopsies and bronchoalveolar lavage (BAL) fluid recovered from patients with asthma have been shown to contain an increased number of activated T cells, B cells, eosinophils and mast cells. Many patients with asthma are sensitized and have specific immunoglogulin E (IgE) antibodies to one or more inhalant allergens. Atopy is, considered to be a major cause of asthma. In atopic individuals, inhalation of allergens preferentially induces a T-helper 2 cell (Th2) response. In the majority of current models, mice are sensitized by itraperitoneal (ip) injection of ovalbumin (OVA), often together with a Th2 skewed adjuvant, such as alum. In the classical mouse model for asthma, C57/BL6 mice are actively sensitized on day 0 by ip injection of 10 μg of OVA absorbed onto 1 mg of alum. From day 14-21 the mice are exposed daily to aerosolized OVA over a 30 minute period. On day 22, airway inflammation is apparent. BAL fluid recovered from these animals demonstrate an increase in peri-bronchiolar space consisting of mixed cellular infiltrates of mononuclear cells and eosinophils. OVA-specific IgE antibodies can be demonstrated in the serum of sensitized animals. The mononuclear cell population consists mainly of cells of Th2 phenotype secreting cytokines IL-4 and IL-5. IL-4 promotes isotype switching of B cells towards IgE synthesis and IL-5 influences the production, maturation and activation of eosinophils.
PAH
The compounds of formula I can be tested in the dog model of pulmonary hypertension as described in Gust, R and Schuster, D. P. Experimental Lung Research, 27:1-12, 2001. They can also be tested in a rabbit model of monocrotaline induced pulmonary hypertension. The compounds of formula I can also be tested in humans with pulmonary arterial hypertension. The effect of the compounds of formula I can be tested in humans with pulmonary arterial hypertension by measurement of its acute effects on cardiopulmonary hemodynamics. The effect of the compounds on right ventricular pressures, pulmonary artery pressures, pulmonary vascular resistance, and cardiac output may be determined. The effect of the compounds on the six minute walk time, and maximal oxygen consumption may be determined in humans with PAH. The effect of the compounds on quality of life (as measured by a questionnaire), hospitalization, and survival may be determined in humans with PAH. In humans PAH may be caused by genetic abnormalities (i.e., primary or familial PAH) or secondary causes such as scleroderma, uncorrected congenital heart disease, mixed collagen vascular disorder, hepatitis C, or other liver disease, HIV infection, or hereditary hemorrhagic teleangiectasia. The effect of the compounds may also be tested on human endothelial cells, fibroblasts and/or smooth muscle cell lines: for example, determination of IC50 for STAT3 phosphorylation in human pulmonary artery smooth muscle cell lines. Cell lines from other species, i.e., the rat may also be examined. The effect of the compounds on precontracted vascular rings from human blood vessels, or blood vessels from other species, i.e., the rat, may be examined. For example, rat pulmonary artery rings preconstricted with phenylephrine, or endothelin, or serotonin, or vasopressin, angiotensin II, or KCL may be studied to determine the dose response to the compounds for vasorelaxation. Other vasoconstrictors may be examined.
The effect of the compounds on hypoxia induced pulmonary vasoconstriction may be examined. A model of hypoxia induced pulmonary hypertension might include study of rats, such as the Fawn-Hooded rat exposed to low oxygen (i.e., 5 percent oxygen). Another model of hypoxia induced pulmonary hypertension might include the fetal calf maintained in a high altitude chamber.
The effect of the compounds may be examined in transgenic models of pulmonary hypertension: i.e., the BMPR2 knockout mouse treated with IL6, the caveolin1 knock out mouse, or the vasoactive intestinal peptide knockout mouse.
The effect of the compounds on histopathologic changes that occur in both human and animal models of PAH may be measured. For example, the compounds may decrease the extent of plexiform lesions in the pulmonary arterioles of diseased lungs. The plexiform lesion consists of endothelial cells, smooth muscle cells, and fibroblasts which proliferate and obstruct to a varying degree, the pulmonary arteriolar lumen.
To assess the activity of small molecule inhibitors of JAK2 an assay has been developed to quantify the activity of the JAK-STAT pathway by measuring the phosphorylation status of the downstream protein STAT5. After ligand binding, a haemopoietic cytokine receptor undergoes conformational change activating associated JAK2 protein. Activated JAK2 then phosphorylates the intracellular portion of the receptor forming binding sites for the recruitment of intracellular signaling proteins. STAT5 is one protein that is recruited to the activated cytokine receptor complex, where it is phosphorylated and then translocates to the nucleus to regulate the expression of a suite of genes that mediate cellular growth and differentiation.
Intracellular flow cytometry can be used to measure tyrosine phosphorylated STAT5 (pYSTAT5) in specific cell populations by gating on lineage-specific haemopoietic surface markers. This is particularly important for JAK2 V617F positive myeloproliferative disease as the clone containing the mutation only forms a variable fraction of all haemopoietic cells within the bone marrow. Erythroid cells have been selected for examination in this study as this lineage is hyperplastic in PV.
Methods
Bone marrow was collected from the ileal crest of patients with JAK2 V617F positive myeloproliferative disease. Flow cytometry assays were performed on fresh bone marrow samples on the day of the biopsy procedure. Bone marrow mononuclear cells were collected by density gradient centrifugation and then 0.75-1.0×106 cells were incubated with compound 3 at various concentrations for one hour in indicator-free RPMI at 37° C. Cells were maximally stimulated with erythropoietin for 10 minutes and then fixed by adding 4% formaldehyde directly into the culture medium. Cells were then permeabilised by cold methanol and then optimal concentrations of fluorescent-labeled antibodies added. Erythroid cells were selected for measurement of pYSTAT5 based on cell surface protein expression (CD45lo, CD71hi population).
Results
Compound 3 was tested in the erythroid cell population at varying concentrations from 3 μM to 0.0041 μM. The first bone marrow specimen was examined with a concentration range of inhibitors from 3 μM to 0.037 μM. The next two patient specimens were examined with a concentration range of between 1 μM and 0.0041 μM.
Unstimulated bone marrow samples with no inhibitor (
All patient samples demonstrated a dose-dependent reduction in STAT5 phosphorylation with increasing dose of inhibitor. Results of flow cytometry experiments are presented in two different formats (
The second format of presentation is presented in the right panel of
All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
This application is a continuation of copending U.S. Ser. No. 12/530,610 filed 19 Mar. 2010, now allowed, which is the national phase of PCT Application PCT/AU2008/000339 having an international filing date of 12 Mar. 2008, and claims priority from U.S. Ser. No. 60/894,264 filed 12 Mar. 2007 and U.S. Ser. No. 61/016,252 filed 21 Dec. 2007.
| Number | Date | Country | |
|---|---|---|---|
| 61016252 | Dec 2007 | US | |
| 60894264 | Mar 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13913362 | Jun 2013 | US |
| Child | 14960710 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12530610 | Mar 2010 | US |
| Child | 13913362 | US |
