Pyrazole derivative calcium release-activated calcium channel modulators and methods of treating non-small cell lung cancer

文档序号：1667307 发布日期：2019-12-31 浏览：46次中文

阅读说明：本技术 吡唑衍生物钙释放激活钙通道调节剂及非小细胞肺癌的治疗方法 (Pyrazole derivative calcium release-activated calcium channel modulators and methods of treating non-small cell lung cancer ) 是由买雅潘·木特胡帕拉尼亚潘斯里坎坦·维斯瓦纳德哈坎西克兰·Vs·瓦拉纳西伽雅特里·斯瓦鲁普于 2010-10-07 设计创作，主要内容包括：本申请涉及吡唑衍生物钙释放激活钙通道调节剂及非小细胞肺癌的治疗方法。公开了新型钙释放激活钙(CRAC)通道抑制剂、其制备方法、含有其的药物组合物和使用其的治疗方法。本公开还涉及用CRAC抑制剂治疗非小细胞肺癌(NSCLC)的方法,并且涉及鉴定用于治疗和诊断癌症的治疗剂的方法。(The present application relates to pyrazole derivatives calcium release-activated calcium channel modulators and methods of treatment of non-small cell lung cancer. Novel calcium release-activated calcium (CRAC) channel inhibitors, methods of their preparation, pharmaceutical compositions containing them, and methods of treatment using them are disclosed. The disclosure also relates to methods of treating non-small cell lung cancer (NSCLC) with CRAC inhibitors, and to methods of identifying therapeutic agents for treating and diagnosing cancer.)

1. A compound of the formula

Or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein

Representation by Ring Hy

Optionally substituted with R' ";

R¹and R²Are the same or different and are independently selected from CH₃、CH₂F、CHF₂、CF₃Substituted or unsubstituted C_(3-5)Cycloalkyl radical, CH₂-OR^a、CH₂-NR^aR^bCN and COOH, with the proviso that:

a)R¹and R²Do not simultaneously represent CF₃；

b)R¹And R²Do not simultaneously represent CH₃；

c) When R is¹Is CF₃When then R is²Is not CH₃(ii) a And

d) when R is¹Is CH₃When then R is²Is not CF₃；

Ring Ar represents:

t, U, V and W are the same or different and are independently selected from CR^aAnd N;

Z¹、Z²and Z³Are the same or different and are independently selected from CR^a、CR^aR^bO, S and-NR^aProvided that Z is¹、Z²And Z³At least one of represents O, S or-NR^a；

L₁And L₂Together represent-NH-C (═ X) -, -NH-S (═ O)_q-, -C (═ X) NH-or-S (═ O)_qNH-or-NH-CR' R "-;

a is absent or selected from- (CR' R ") -, O, S (═ O)_qC (═ X) and-NR^a；

R 'and R' are the same OR different and are independently selected from hydrogen, hydroxy, cyano, halogen, -OR^a、-COOR^a、-S(＝O)_q-R^a、-NR^aR^b、-C(＝X)-R^aSubstituted or unsubstituted C_(1-6)Alkyl radical, substituted or unsubstituted C_(1-6)Alkenyl, substituted or unsubstituted C_(1-6)Alkynyl and substituted or unsubstituted C_(3-5)Cycloalkyl, or R 'and R' are linkableInto a substituted or unsubstituted, saturated or unsaturated 3-to 6-membered ring, which ring may optionally comprise one or more rings which may be the same or different and are selected from O, NR^aAnd a heteroatom of S;

r' "is selected from hydrogen, hydroxy, cyano, halogen, -OR^a、-COOR^a、-S(＝O)_q-R^a、-NR^aR^b、-C(＝X)-R^aSubstituted or unsubstituted C_(1-6)Alkyl radical, substituted or unsubstituted C_(1-6)Alkenyl, substituted or unsubstituted C_(1-6)Alkynyl and substituted or unsubstituted C_(3-5)A cycloalkyl group;

x at each occurrence is independently selected from O, S and-NR^a；

Cy is a bicyclic ring selected from the group consisting of substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclyl groups, substituted or unsubstituted aryl groups, and substituted or unsubstituted heteroaryl groups;

each occurrence of R^aAnd R^bIdentical OR different and independently selected from hydrogen, nitro, hydroxy, cyano, halogen, -OR^c、-S(＝O)_q-R^c、-NR^cR^d、-C(＝Y)-R^c、-CR^cR^d-C(＝Y)-R^c、-CR^cR^d-Y-CR^cR^d-、-C(＝Y)-NR^cR^d-、-NRR^d-C(＝Y)-NR^cR^d-、-S(＝O)_q-NR^cR^d-、-NR^cR^d-S(＝O)_q-NR^cR^d-、-NR^cR^d-NR^cR^d-, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroarylalkyl, orWhen R is^aAnd R^bWhen taken directly in combination with the same atom, they may be linked to form a substituted or unsubstituted saturated or unsaturated 3-to 10-membered ring, which may optionally include one or more ring members which may be the same or different and selected from O, NR^cAnd a heteroatom of S;

each occurrence of R^cAnd R^dIdentical or different and are independently selected from the group consisting of hydrogen, nitro, hydroxy, cyano, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heterocyclylalkyl, or when two R's are present^cAnd/or R^dWhen the substituents are directly bound to the same atom, they may be linked to form a substituted or unsubstituted saturated or unsaturated 3-to 10-membered ring, which may optionally include one or more heteroatoms, which may be the same or different, selected from O, NH and S;

each occurrence of Y is selected from the group consisting of O, S and-NR^a(ii) a And is

Q for each occurrence independently represents 0, 1 or 2;

with the proviso that (e) the compound of formula (I) is not:

n- [4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl ] -1-methyl-3- (trifluoromethyl) -1H-thieno [2, 3-c ] pyrazole-5-carboxamide or N- [4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl ] -pyrazolo [1, 5-a ] pyrimidine-2-carboxamide.

2. The compound of claim 1, wherein Hy is

3. The compound of claim 2, wherein Hy is

4. The compound of claim 3, wherein Hy is

5. The compound of claim 1, wherein Ar is

6. The compound of claim 6, wherein Ar is

7. The compound of claim 1, wherein L₁And L₂Together represent-NH-C (═ O) -, -NH-S (═ O)_q-, -C (═ O) NH-or-NH-CH₂-。

8. The compound of claim 7, wherein L₁And L₂Together represent-NH-C (═ O) -, -C (═ O) NH-, or S (═ O)_qNH-、-NH-CH₂-。

9. The compound of claim 1, wherein a is absent or selected from- (CR' R ") -or-NR^a。

10. The compound of claim 9, wherein a is-CH₂-or-CHMe-, - (CR 'R') -, wherein R 'and R' are joined to form a substituted or unsubstituted saturated or unsaturated 3-6 membered ring, which ring may optionally comprise one or more rings which may be the same or different and are selected from O, C, O,NR^aand a heteroatom of S.

Technical Field

The present invention relates to Calcium Release Activated Calcium (CRAC) channel inhibitors of formula I and pharmaceutically acceptable salts thereof, processes for their preparation, pharmaceutical compositions containing them and methods of treatment using them.

The invention also relates to methods of treating non-small cell lung cancer (NSCLC) with CRAC inhibitors and methods of identifying therapeutic agents for treating and diagnosing cancer.

Background

The regulation of intracellular calcium is a key element of signal transduction into and within cells. Cellular responses to growth factors, neurotransmitters, hormones and a variety of other signaling molecules are initiated through a calcium-dependent process. The importance of calcium ions as a second messenger is underscored by a number of different mechanisms that work together to maintain calcium homeostasis. Changes in intracellular free calcium ion concentration represent the most widespread and important signaling events that regulate numerous cellular responses. The broad route of calcium entry into cells is through the calcium reservoir-operated channel (SOC), i.e. many cell types employ a calcium reservoir-operated calcium entry as the primary pathway for calcium influx. This mechanism is used after calcium ions are released from the calcium pool, where the pool emptying results in activation of Calcium Release Activated Calcium (CRAC) channels.

The subfamily CRAC channels of the store-operated channel are activated by the release of calcium from the intracellular calcium store, particularly from the Endoplasmic Reticulum (ER). These pathways are key factors in the regulation of a wide range of cellular functions, including muscle contraction, protein and fluid secretion, and control of cell growth and proliferation, and thus play a fundamental role in various diseases (e.g., immune disorders) and allergies. Among the several biophysically distinct reservoir steering currents, one of the most characteristic and selective to calcium ions is the CRAC current. Thus, CRAC channels mediate essential functions from secretion to gene expression and cell growth and form the network necessary for immune cell activation to establish an adaptive immune response. Recently, two proteins, matrix interactive molecule (STIM1) and CRAC modulator 1(CRACM1 or Orai1), have been identified as essential components for the complete reconstitution and enhancement of CRAC currents in heterologous expression systems with similar biophysical fingerprints. In mammals, there are several homologs of these proteins: STIM1 and STIM2 in the endoplasmic reticulum and CRACM1, CRACM2 and CRACM3 in the plasma membrane.

CRAC currents were initially found in lymphocytes and mast cells, and have been characterized simultaneously in various cell lines, such as S2 drosophila, DT 40B cells, hepatocytes, dendritic cells, megakaryocytes, and canine kidney epithelial cells. In lymphocytes and mast cells, activation by an antigen or Fc receptor triggers the initiation of the second messenger (1, 4, 5) -inositol triphosphate (Ins (1, 4, 5) P₃) The resulting release of calcium ions from the intracellular calcium pool, which in turn leads to influx of calcium ions through the CRAC channels in the plasma membrane. Calcium pool-operated Ca characterized in smooth muscle, A431 epidermal cells, endothelial cells and prostate cancer cell lines from various tissues²⁺The current showed a change in biophysical characteristics, indicating that the source of the molecule was different.

For example, calcium ions that flow through the cell membrane are important in lymphocyte activation and adaptive immune responses. It has been demonstrated that [ Ca ] is caused by TCR (T cell antigen receptor) stimulation²⁺]Oscillations were significant and appeared to involve only a single calcium ion flow pathway, the cisterna-operated CRAC channel. See, for example, Lewis "Calcium signalling mechanisms in Tlympcytes," Annu. Rev. Immunol.19, (2001), 497-521; feske et al "Ca⁺⁺calceinin signalling in cells of the immune system, "biochem. biophysis. Res. Commun.311, (2003), 1117-1132; hogan et al, "transformation regulation by calcium, calceinin, and NFAT," Genes Dev.17, (2003) 2205-.

It has been confirmed that intracellular calcium plays an important role in various cellular functions, and its concentration is regulated by calcium ions flowing through calcium channels on cell membranes. Calcium ion channels located in the nervous, endocrine, cardiovascular and skeletal systems and regulated by membrane potential are called voltage-manipulated Ca²⁺(VOC) channels. These channels are divided into L, N, P, Q, R and T subtypes. Excess Ca flowing through VOC channels²⁺Causing hypertension and brain dysfunction. In contrast, regardless of membrane potential, calcium ion channels on inflammatory cells (including lymphocytes, mast cells, and neutrophils) can be activated. This type of calcium ion channel has been shown to play a role in the crisis and exacerbation of inflammatory and autoimmune diseases. In T cells, early activation stages from pre-Ca have been reported²⁺Post-neutralization of Ca²⁺And (4) event composition. Stimulation of T cell receptors induces pre-Ca²⁺Events, including generation of IP3, then Ca²⁺Is released from the Endoplasmic Reticulum (ER). After Ca²⁺In the event, Ca is in ER²⁺Emptying induces CRAC channel activation and capacity Ca through CRAC channels²⁺Flow maintenance of intracellular high Ca²⁺Concentration ([ Ca ]²⁺]i) In that respect Thus extended height [ Ca2+]i activate cytosolic signaling to generate lipid mediators (e.g., LTD)₄) Cytokines [ e.g., interleukin-2 (IL-2) ]]And matrix metalloproteinases, which are involved in the pathogenesis of inflammatory and autoimmune diseases.

These facts indicate that CRAC channel modulators may be useful in the treatment of diseases caused by inflammatory cell activation without the side effects observed in steroids. Because VOC channel modulators will cause adverse events in the nervous and cardiovascular systems, it may be necessary for CRAC channel modulators to exhibit sufficient selectivity for VOC channels if used as anti-inflammatory drugs.

Thus, it is believed that CRAC channel modulators are useful in the treatment, prevention and/or amelioration of calcium release-activated calcium channel-associated diseases or conditions, including, but not limited to, inflammation, glomerulonephritis, uveitis, liver diseases or conditions, kidney diseases or conditions, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, vasculitis, dermatitis, osteoarthritis, inflammatory muscle diseases, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allo-or xeno-transplantation, transplant rejection, graft-versus-host disease, lupus erythematosus, type I diabetes, pulmonary fibrosis, dermatomyositis, thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, hepatitis, and atopic dermatitis, Asthma, sjogren's syndrome, cancer and proliferative diseases, as well as autoimmune diseases or disorders. See, for example, international publication nos. WO 2005/009954, WO 2005/009539, WO 2005/009954, WO 2006/034402, WO 2006/081389, WO 2006/081391, WO 2007/087429, WO 2007/087427, WO2007087441, WO 200/7087442, WO 2007/087443, WO 2007/089904, WO 2007109362, WO2007/112093, WO 2008/039520, WO 2008/063504, WO 2008/103310, WO 2009/017818, WO2009/017819, WO 2009/017831, WO 2010/039238, WO 2010/039237, WO2010/039236, WO2009/089305, and WO 2009/038775 and U.S. publication nos.: US 2006/0173006 and US 2007/0249051.

CRAC channel inhibitors that have been identified include SK & F96365 (1), econazole (2), and L-651582 (3).

However, these molecules lack sufficient potency and selectivity for VOC channels and are therefore unsuitable for therapeutic use.

Recent publications by Taiji et al (European Journal of Pharmacology, 560, 225-.

Yasurio Yonneky et al disclose YM-58483 that is selective for CRAC channels in voltage-operated channels (VOCs) with a selectivity index of 31.

Other CRAC channel modulators are disclosed including various biaryl and/or heterocyclic formanilide compounds, including, for example, PCT or US patent applications assigned to Synta Pharmaceuticals, namely WO 2005/009954, WO 2005/009539, WO 2005/009954, WO 2006/034402, WO 2006/081389, WO 2006/081391, WO 2007/087429, WO 2007/087427, WO2007087441, WO 200/7087442, WO 2007/087443, WO 2007/089904, WO 2007109362, WO2007/112093, WO 2008/039520, WO 2008/063504, WO 2008/103310, WO 2009/017818, WO2009/017819, WO 2009/017831, WO 2010/039238, WO 2010/039237, WO2010/039236, WO2009/089305 and WO 2009/038775, US 2006/0173006 and US 2007/0249051.

Other patent publications relating to CRAC channel modulators include astella, Queens Medical Centre, Calcimedica and other applications, namely WO 2007/121186, WO 2006/050214, WO 2007/139926, WO2008/148108, US 7,452,675, US 2009/023177, WO 2007/139926, US6,696,267, US6,348,480, WO2008/106731, US 2008/0293092, WO 2010/048559, WO 2010/027875, WO2010/025295, WO 2010/034011, WO2010/034003, WO 2009/076454, WO 2009/035818, US 2010/0152241, US 2010/0087415, US 2009/0311720 and WO 2004/078995.

Further reviews and literature disclosures in the field of CRAC channels include Isabella Derler et al, Expert Opinion in Drug Discovery, 3(7), 787-; young G et al, Cell Calcium, 42, 145-156, 2007; yasurio Yonetoky et al, Bio. & med. chem., 14, 4750-; and Yasurio Yonetoky et al, Bio. & med. chem., 14, 5370-. All of these patents and/or patent applications and literature publications are incorporated herein by reference for all purposes.

Cancer is a major public health problem in india, the united states and many other regions of the world. Now, death in india 1/4 is due to cancer. Lung cancer is the leading cause of cancer death worldwide due to its high incidence and mortality, with a 5-year survival rate estimated to be-10% for non-small cell lung cancer (NSCLC). Further studies on the tumorigenesis and chemoresistance machinery of lung Cancer have been reported to be required to improve survival (Jemal A et al, Cancer statics, CACACANCER.J.Clin., 56, 106-. There are 4 major types of NSCLC, namely adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma, and large cell carcinoma. Based on cellular morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC (Travis et al, Lung Cancer Principles and Practice, Lippincott-Raven, New York, 361-395, 1996). Adenocarcinomas are characterized by more peripheral locations in the lung and often have K-ras oncogene mutations (Gazdar et al, Anticancer Res., 14, 261-267, 1994). Squamous cell carcinomas are usually more central and often carry a p53 gene mutation (Niklinska et al, Folia histochem. cytobiol., 39, 147-148, 2001).

Most NSCLC are characterized by the presence of ras mutations, rendering the patient relatively insensitive to treatment by known kinase inhibitors. Thus, current lung cancer treatments are generally limited by cytotoxic drugs, surgery, and radiation therapy. There is a need for treatments that have fewer side effects and that target cancer cells more specifically, are less invasive, and improve patient prognosis.

The identification of lung tumor initiating cells and associated markers can be used to optimize treatment methods and predictive and prognostic information for lung cancer patients. Thus, there remains a need for new methods of predicting, evaluating, and treating patients afflicted with lung cancer.

There remains an unmet and urgent need for small molecule modulators specific for Stim1 and/or Orai1 to modulate and/or modulate the activity of CRAC channels, particularly for the treatment of CRAC-related diseases and disorders.

Disclosure of Invention

The present invention relates to compounds of formula (I) and processes for their preparation, pharmaceutical compositions containing them and methods of treatment using them.

In particular, it relates to compounds of formula (I) and pharmaceutically acceptable salts thereof, which are calcium release-activated calcium channel modulators useful for the treatment, prevention, inhibition and/or amelioration of calcium release-activated calcium channel-related diseases or disorders.

In one aspect, the invention relates to compounds of formula (I):

or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein

Representation by Ring Hy

Optionally substituted with R' ";

a)R¹and R²Do not simultaneously represent CF₃；

b)R¹And R²Do not simultaneously represent CH₃；

c) When R is¹Is CF₃When then R is²Is not CH₃(ii) a And

d) when R is¹Is CH₃When then R is²Is not CF₃；

Ring Ar represents:

t, U, V and W are the same or different and are independently selected from CR^aAnd N;

Z¹、Z²and Z³Are the same or different and are independently selected from CR^a、CR^aR^bO, S and-NR^aProvided that Z is¹、Z²And Z³At least one of represents O, S or-NR^a；

L₁And L₂Together represent-NH-C (═ X) -, -NH-S (═ O)_q-, -C (═ X) NH-or-S (═ O)_qNH-or-NH-CR' R "-;

a is absent or selected from- (CR' R ") -, O, S (═ O)_qC (═ X) and-NR^a；

R 'and R' are the same OR different and are independently selected from hydrogen, hydroxy, cyano, halogen, -OR^a、-COOR^a、-S(＝O)_q-R^a、-NR^aR^b、-C(＝X)-R^aSubstituted or unsubstituted C_(1-6)Alkyl radical, substituted or unsubstituted C_(1-6)Alkenyl, substituted or unsubstituted C_(1-6)Alkynyl and substituted or unsubstituted C_(3-5)Cycloalkyl, or R 'and R' may be joined to form a substituted or unsubstituted saturated or unsaturated 3-6 membered ring, which ring may optionally include one or more rings which may be the same or different and selected from O, NR^aAnd a heteroatom of S;

X at each occurrence is independently selected from O, S and-NR^a；

each occurrence of R^aAnd R^bIdentical OR different and independently selected from hydrogen, nitro, hydroxy, cyano, halogen, -OR^c、-S(＝O)_q-R^c、-NR^cR^d、-C(＝Y)-R^c、-CR^cR^d-C(＝Y)-R^c、-CR^cR^d-Y-CR^cR^d-、-C(＝Y)-NR^cR^d-、-NRR^d-C(＝Y)-NR^cR^d-、-S(＝O)_q-NR^cR^d-、-NR^cR^d-S(＝O)_q-NR^cR^d-、-NR^cR^d-NR^cR^d-, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroarylalkyl, or when R is^aAnd R^bWhen taken directly in combination with the same atom, they may be linked to form a substituted or unsubstituted saturated or unsaturated 3-to 10-membered ring, which may optionally include one or more ring members which may be the same or different and selected from O, NR^cAnd a heteroatom of S;

each occurrence of Y is selected from the group consisting of O, S and-NR^a(ii) a And is

Q for each occurrence independently represents 0, 1 or 2;

with the proviso that (e) the compound of formula (I) is not:

In a preferred embodiment, R¹Is cyclopropyl.

In a preferred embodiment, R²Is cyclopropyl.

According to a preferred embodiment, Hy is

More preferred are compounds of formula (I) wherein Hy is

According to a preferred embodiment, Ar is

More preferred are compounds of formula (I) wherein Ar is

According to a preferred embodiment, L₁And L₂Together represent-NH-C (═ O) -、-NH-S(＝O)_q-, -C (═ O) NH-or-NH-CH₂-。

According to a preferred embodiment, a is absent or selected from- (CR' R ") -, O, S (═ O)_qC (═ X) and-NR^a. More preferably, A is-CH₂-, -CHMe-or- (CR 'R') -, wherein R 'and R' are joined to form a substituted or unsubstituted saturated or unsaturated 3-6 membered ring, which ring may optionally comprise one or more rings which may be the same or different and are selected from O, NR^a(e.g., NH) and S.

More preferred are compounds of formula (I) wherein A is

More preferred are compounds of formula (I) wherein a is absent.

More preferred are compounds of formula (I) wherein A is-CH₂-。

According to a preferred embodiment, Cy is

More preferred are compounds of formula (I) wherein Cy is

Yet another embodiment is a compound having formula (IA):

or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein the variables (e.g., R' ", R¹、R²、T、U、V、W、L₁、L₂A and Cy) are as defined above for formula (I), with the proviso that the compound of formula (IA) is not any of the compounds of conditions (a-e) defined above.

Yet another embodiment is a compound having the formula (IA-I):

or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein the variables (e.g., R' ", R¹、R²T, U, V, W, A and Cy) are as defined above for formula (I), with the proviso that the compound of formula (IA) is not any of the compounds of conditions (a-e) defined above.

More preferred are compounds of formula (IA-I)

Or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein

R¹And R²Are the same or different and are independently selected from CH₂F、CHF₂、CF₃And cyclopropyl, with the proviso that R¹And R²Do not simultaneously represent CF₃；

R' "is hydrogen or halogen;

t, U, V, W is independently CR^aOr N;

R^ais hydrogen or halogen;

a is absent or selected from

And is

Cy is selected from substituted or unsubstituted bicyclic aryl or substituted or unsubstituted bicyclic heteroaryl,

with the proviso that the compound of formula (IA) is not any compound of condition (e) as defined above.

More preferred are compounds of formula (IA-I) wherein R¹And R²Both represent cyclopropyl.

More preferred are compounds of formula (IA-I) wherein R¹And R²Is CF₃And the other is cyclopropyl.

More preferred are compounds of formula (IA-I) wherein R¹Is cyclopropyl and R²Is CF₃。

More preferred are compounds of formula (IA-I) wherein T, U, V, W is CH, CF or N.

More preferred are compounds of formula (IA-I) wherein T is CF or N, and each of U, V and W is CH.

More preferred are compounds of formula (IA-I) wherein each of T and V is independently CF or N and each of U and W is CH.

More preferred are compounds of formula (IA-I) wherein A is absent or selected from

More preferred are compounds of formula (IA-I) wherein Cy is selected from

Yet another embodiment are compounds having formula (IA-II)

Or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof, wherein

R¹And R²Are the same or different and are independently selected from CH₂F、CHF₂、CF₃And cyclopropyl, with the proviso that R¹And R²Do not simultaneously represent CF₃；

R' "is hydrogen or halogen;

t, U, V, W is independently CR^aOr N;

R^ais hydrogen or halogen;

a is absent or selected from

And is

Cy is selected from substituted or unsubstituted C_(8-13)A bicyclic heteroaryl group,

with the proviso that the compound of formula (IA) is not any compound of condition (e) as defined above.

More preferred are compounds of formula (IA-II) wherein R¹And R²Both represent cyclopropyl.

More preferred are compounds of formula (IA-II) wherein R¹And R²Is CF₃And the other is cyclopropyl.

More preferred are compounds of formula (IA-II) wherein R¹Is cyclopropyl and R²Is CF₃。

More preferred are compounds of formula (IA-II) wherein T, U, V, W is CH, CF or N.

More preferred are compounds of formula (IA-II) wherein T is CF or N, and each of U, V and W is CH.

More preferred are compounds of formula (IA-II) wherein each of T and V is independently CF or N and each of U and W is CH.

More preferred are compounds of formula (IA-II) wherein A is absent or is-CH₂-。

In one embodiment, A is-CH₂-。

More preferred are compounds of formula (IA-II) wherein Cy is selected from

Yet another embodiment are compounds having formula (IA-III)

Or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof,

wherein

R¹And R²Are the same or different and are independently selected from CH₂F、CHF₂、CF₃Cyclopropyl, provided that R is¹And R²Do not simultaneously represent CF₃；

T and V are the same or different and are independently selected from CF and N;

each of U and V is CR^a；

L₁And L₂Together represent-NH-C (═ X) -, -NH-S (═ O)_q-, -C (═ X) NH-or-S (═ O)_qNH-or-NH-CR' R "-;

a is absent or selected from- (CR 'R') -and-NR^a；

R 'and R' in each occurrence are the same or different and are independently selected from hydrogen or substituted or unsubstituted C_(1-6)The alkyl group, or R 'and R' may be joined to form a substituted or unsubstituted saturated or unsaturated 3-6 membered ring, which ring may optionally include one or more rings which may be the same or different and selected from O, NR^aAnd a heteroatom of S;

r' "is selected from hydrogen or halogen;

x at each occurrence is independently selected from O, S and-NR^a；

Cy is a bicyclic ring selected from the group consisting of substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

each occurrence of R^aAnd R^bIdentical OR different and independently selected from hydrogen, nitro, hydroxy, cyano, halogen, -OR^c、-S(＝O)_q-R^c、-NR^cR^d、-C(＝Y)-R^c、-CR^cR^d-C(＝Y)-R^c、-CR^cR^d-Y-CR^cR^d-、-C(＝Y)-NR^cR^d-、-NRR^d-C(＝Y)-NR^cR^d-、-S(＝O)_q-NR^cR^d-、-NR^cR^d-S(＝O)_q-NR^cR^d-、-NR^cR^d-NR^cR^d-, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroarylalkyl, or when R is^aAnd R^bWhen the substituents are bonded directly to the same atom, they may be linked to form a substituted or unsubstituted saturated or unsaturated 3-to 10-membered ring, which may optionally include one or more groups selected from O, NR which may be the same or different^cAnd a heteroatom of S;

each occurrence of R^cAnd R^dIdentical or different and are independently selected from the group consisting of hydrogen, nitro, hydroxy, cyano, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heterocyclylalkyl, or when two R's are present^cAnd/or R^dWhen substituents are bound directly to the same atom, they may be linked to form a saturated or unsubstituted moietyAn unsaturated 3-to 10-membered ring, which ring may optionally include one or more heteroatoms, which may be the same or different, selected from O, NH and S;

each occurrence of Y is selected from the group consisting of O, S and-NR^a(ii) a And is

Q for each occurrence independently represents 0, 1 or 2.

More preferred are compounds of formula (IA-III) wherein R¹And R²Both represent cyclopropyl.

More preferred are compounds of formula (IA-III) wherein R¹And R²Is CF₃And the other is cyclopropyl.

More preferred are compounds of formula (IA-III) wherein R¹And R²Is CF₃And the other is CH₂F、CHF₂。

More preferred are compounds of formula (IA-III) wherein R¹Is cyclopropyl and R²Is CF₃。

More preferred are compounds of formula (IA-III) wherein T is CF or N.

More preferred are compounds of formula (IA-III) wherein U, V, W is CH, CF or N.

More preferred are compounds of formula (IA-III) wherein L₁And L₂Together represent-NH-C (═ O) -, C (═ O) NH-, or-NH-CH₂-。

More preferred are compounds of formula (IA-III) wherein A is absent or is-NH-or-CH₂-。

More preferred are compounds of formula (IA-III) wherein Cy is selected from

Yet another embodiment are compounds having formula (IA-IV)

Or a tautomer, prodrug, N-oxide, pharmaceutically acceptable ester, or pharmaceutically acceptable salt thereof,

wherein

R¹And R²Are the same or different and are independently selected from CH₂F、CHF₂、CF₃Cyclopropyl, provided that R is¹And R²Do not simultaneously represent CF₃；

T and V are the same or different and are independently selected from CH, CF and N;

each of U and V is CR^a；

L₁And L₂Together represent-NH-C (═ X) -, -NH-S (═ O)_q-, -C (═ X) NH-or-S (═ O)_qNH-or-NH-CR' R "-;

a is selected from the group consisting of- (CR 'R') -and-NR^a；

r' "is selected from hydrogen or halogen;

x at each occurrence is independently selected from O, S and-NR^a；

Cy is a substituted or unsubstituted bicyclic heterocyclic group;

each occurrence of R^aAnd R^bIdentical OR different and independently selected from hydrogen, nitro, hydroxy, cyano, halogen, -OR^c、-S(＝O)_q-R^c、-NR^cR^d、-C(＝Y)-R^c、-CR^cR^d-C(＝Y)-R^c、-CR^cR^d-Y-CR^cR^d-、-C(＝Y)-NR^cR^d-、-NRR^d-C(＝Y)-NR^cR^d-、-S(＝O)_q-NR^cR^d-、-NR^cR^d-S(＝O)_q-NR^cR^d-、-NR^cR^d-NR^cR^d-, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl,Substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroarylalkyl, or when two R's are^aAnd R^bWhen the substituents are bonded directly to the same atom, they may be linked to form a substituted or unsubstituted saturated or unsaturated 3-to 10-membered ring, which may optionally include one or more groups selected from O, NR which may be the same or different^cAnd a heteroatom of S;

each occurrence of Y is selected from the group consisting of O, S and-NR^a(ii) a And is

Q for each occurrence independently represents 0, 1 or 2.

More preferred are compounds of formula (IA-IV) wherein R¹And R²Both represent cyclopropyl.

More preferred are compounds of formula (IA-IV) wherein R¹And R²Is CF₃And the other is cyclopropyl.

More preferred are compounds of formula (IA-IV) wherein R¹And R²Is CF₃And the other is CH₂F、CHF₂。

More preferred are compounds of formula (IA-IV) wherein R¹Is cyclopropyl and R²Is CF₃。

More preferred are compounds of formula (IA-IV) wherein T is CH, CF or N.

More preferred are compounds of formula (IA-IV) wherein U, V, W is CH, CF or N.

More preferred are compounds of formula (IA-IV) wherein L₁And L₂Together represent-NH-C (═ O) -, C (═ O) NH-, or-NH-CH₂-。

More preferred are compounds of formula (IA-IV) wherein A is absent or is-NH-or-CH₂-。

More preferred are compounds of formula (IA-IV) wherein Cy is selected from

In yet another embodiment the invention relates to methods of treating non-small cell lung cancer (NSCLC) with Calcium Release Activated Calcium (CRAC) inhibitors and methods of identifying therapeutic agents for the treatment and diagnosis of cancer. In certain embodiments, the CRAC inhibitor is a compound of formula I, IA-II, IA-III, or IA-IV as in any of the embodiments described herein.

The present inventors have found that cancer cells expressing an ORAI (e.g., ORAI1, ORAI2, or ORAI3) or STIM (e.g., STIM1 or STIM2) are sensitive to treatment with calcium release-activated calcium (CRAC) inhibitors. These types of cancer cells are expressed in many patients with non-small cell lung cancer (NSCLC).

One embodiment of the invention is a method of treating a patient suffering from NSCLC by administering to the patient an effective amount of a CRAC inhibitor. In a preferred embodiment, at least some of the cancer cells express ORAI1, STIM1, or STIM 2. CRAC inhibitors may be used as monotherapy or as adjunct therapy to one or more other methods of treating lung cancer (or NSCLC). In certain embodiments, the CRAC inhibitor is a compound of formula I, IA-II, IA-III, or IA-IV as in any of the embodiments described herein.

Another embodiment is a method of treating a patient having NSCLC by altering calcium flow into at least some cancer cells, preferably by enhancing the expression levels of calcium release-activated calcium (CRAC) channels and/or STIM proteins in the plasma membrane of at least some cancer cells.

Yet another embodiment is a method of identifying a candidate agent for treating NSCLC. The method comprises (a) determining whether (i) the candidate agent modulates a Calcium Release Activated Calcium (CRAC) channel, and/or (ii) the candidate agent modulates the expression of Stim protein of a CRAC channel, or both; and (b) selecting the candidate agent according to its ability to modulate the CRAC channel or the Stim protein of the CRAC channel.

In a preferred embodiment, the candidate agent alters the calcium flux into the cancer cell. For example, candidate agents may selectively modulate CRAC channels or STIM proteins. Preferably, the candidate agent selectively inhibits a CRAC channel or STIM protein. For example, the suppressed CRAC channel may be selected from CRACM1/Orai1, CRACM2/Orai2, and CRACM3/Orai 3. In another embodiment, the candidate agent inhibits STIM protein localized on the endoplasmic reticulum membrane of the cell. In particular embodiments, the STIM protein is selected from the transmembrane protein family, such as STIM1 or STIM 2. According to a preferred embodiment, the STIM protein is STIM 1. In certain embodiments, a candidate agent is a compound of formula I, IA-II, IA-III, or IA-IV as in any of the embodiments described herein.

Yet another embodiment is a pharmaceutical composition for treating NSCLC comprising (a) a candidate agent identified according to the above method as effective for treating NSCLC, and (b) a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, a candidate agent is a compound of formula I, IA-II, IA-III, or IA-IV as in any of the embodiments described herein.

Yet another embodiment is a method of treating a patient having NSCLC by administering to the patient (a) an effective amount of a candidate agent identified according to the above method or (b) one or more pharmaceutical compositions comprising (i) a candidate agent identified according to the above method that is effective in treating NSCLC and (b) a pharmaceutically acceptable carrier, diluent or excipient, wherein the total amount of candidate agents provided by the pharmaceutical composition provides a therapeutically effective amount of the candidate agent. In certain embodiments, a candidate agent is a compound of formula I, IA-II, IA-III, or IA-IV as in any of the embodiments described herein.

Yet another embodiment is a method of determining whether a human is predisposed to or afflicted with lung cancer by detecting the levels of Calcium Release Activated Calcium (CRAC) channels and/or STIM proteins in lung cells, such as cancer cells. In one embodiment, the method comprises detecting elevated levels of Orai and/or STIM proteins. For example, the method comprises detecting elevated levels of STIM protein in cancer cells. For example, the STIM proteins to be detected are members of the STIM family of transmembrane proteins, such as STIM1 or STIM 2. In one embodiment, the STIM protein to be detected is STIM 1.

Representative compounds of the invention include the compounds specified below and in table 1, and pharmaceutically acceptable salts thereof. The invention should not be construed as being limited to said compounds.

N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -1H-benzo [ d ] imidazole-6-carboxamide

N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -1H-benzo [ d ] [1, 2, 3] triazole-6-carboxamide

N- [4- (3, 5-Bicyclopropyl-1H-pyrazol-1-yl) phenyl ] quinoline-6-carboxamide hydrochloride

N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] quinoxaline-6-carboxamide

2- (1H-benzo [ d ] imidazol-1-yl) -N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] acetamide

2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] acetamide

N- [4- (3, 5-dicyclopropyl-1H-pyrazol-1-yl) phenyl ] -2- (1H-indol-3-yl) acetamide

N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -2- (imidazo [1, 2-a ] pyridin-2-yl) acetamide hydrochloride

N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -2- (quinolin-6-yl) acetamide:

n- [4- (3, 5-Bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -2- (quinolin-6-yl) acetamide hydrochloride

11.2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- (4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) -3-fluorophenyl) acetamide

N- [4- (3, 5-Bicyclopropyl-1H-pyrazol-1-yl) -3-fluorophenyl ] -2- (quinolin-6-yl) acetamide hydrochloride

N- [6- (3, 5-dicyclopropyl-1H-pyrazol-1-yl) pyridin-3-yl ] quinoline-6-carboxamide dihydrochloride

N- [6- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) pyridin-3-yl ] quinoxaline-6-carboxamide

2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- [6- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) pyridin-3-yl ] acetamide

N- [6- (3, 5-Bicyclopropyl-1H-pyrazol-1-yl) pyridin-3-yl ] -2- (quinolin-6-yl) acetamide dihydrochloride

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } quinoline-6-carboxamide hydrochloride

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } quinoxaline-6-carboxamide

19.2- (1H-benzo [ d ] imidazol-1-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } acetamide

2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } acetamide

2- (2H-benzo [ d ] [1, 2, 3] triazol-2-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } acetamide

2- (3H- [1, 2, 3] triazolo [4, 5-b ] pyridin-3-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } acetamide

(S) -2- (3H- [1, 2, 3] triazolo [4, 5-b ] pyridin-3-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } propanamide

24.2- (6-amino-9H-purin-9-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } acetamide

N- (4- (5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl) phenyl) -2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetamide

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl) phenyl) -2- (imidazo [1, 2-a ] pyridin-2-yl) acetamide hydrochloride

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } -2- (quinolin-6-yl) acetamide hydrochloride

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] phenyl } -2- (quinolin-6-yl) propanamide hydrochloride

N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] -3-fluorophenyl } -1H-benzo [ d ] [1, 2, 3] triazole-6-carboxamide

2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] -3-fluorophenyl } acetamide

N- {6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] pyridin-3-yl } -1H-benzo [ d ] [1, 2, 3] triazole-5-carboxamide

2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- {6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] pyridin-3-yl } acetamide

33.2- (2H-benzo [ d ] [1, 2, 3] triazol-2-yl) -N- {6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] pyridin-3-yl } acetamide

N- {6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] pyridin-3-yl } -2- (quinolin-6-yl) acetamide hydrochloride

35.2- (1H-benzo [ d ] [1, 2, 3] triazol-1-yl) -N- {6- [ 4-chloro-5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] pyridin-3-yl } acetamide

36.4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl ] -3-fluoro-N- (quinolin-6-ylmethyl) benzamide hydrochloride

37.1- [4- (3, 5-Bicyclopropyl-1H-pyrazol-1-yl) phenyl ] -3- (quinolin-6-yl) urea

TABLE 1

Compounds of the invention (e.g., compounds of formula I, IA-I, IA-II, IA-III, and/or IA-IV, including pharmaceutically acceptable esters and salts thereof) are useful for the treatment, prevention, inhibition, and/or amelioration of diseases and disorders associated with Calcium Release Activated Calcium (CRAC) channels.

Another embodiment of the invention is a method of treating a disease or disorder by modulating a CRAC channel by administering to a patient in need of such treatment an effective amount of a compound of the invention (e.g., a compound of formula I, IA-I, IA-II, IA-III and/or IA-IV as defined above).

Yet another embodiment of the invention is a method of treating a disease or disorder by modulating the CRAC pathway by administering to a patient in need of such treatment an effective amount of a compound of the invention (e.g., a compound of formula I, IA-I, IA-II, IA-III, and/or IA-IV as defined above) in combination with (simultaneously or sequentially) at least one other anti-inflammatory agent.

Yet another embodiment of the invention is a method of treating a disease or disorder by modulating the CRAC pathway by administering to a patient in need of such treatment an effective amount of a compound of the invention (e.g., a compound of formula I, IA-I, IA-II, IA-III, and/or IA-IV as defined above) in combination (simultaneously or sequentially) with at least one other anti-cancer agent.

The compounds of the invention inhibit the entry of reservoir-manipulated calcium, block the assembly of SOCE units, alter the functional interaction of proteins that form the reservoir-manipulated calcium channel complex and alter the functional interaction of STIM1 with Orai 1. These compounds are SOC channel pore blockers and are CRAC channel pore blockers.

Modulation of intracellular calcium by the compounds described herein and for the treatment of diseases, disorders, or conditions in which modulation of intracellular calcium has a beneficial effect. In one embodiment, the compounds described herein inhibit calcium pool-operated calcium entry. In one embodiment, a compound of the invention capable of modulating intracellular calcium levels blocks the assembly of SOCE units. In another embodiment, a compound of the invention capable of modulating intracellular calcium levels alters the functional interactions of proteins that form the pool-operated calcium channel complex. In one embodiment, a compound of the invention capable of modulating intracellular calcium levels alters the functional interaction of STIM1 with Orai 1. In other embodiments, the compounds of the invention capable of modulating intracellular calcium levels are SOC channel pore blockers. In other embodiments, the compounds of the invention capable of modulating intracellular calcium levels are CRAC channel pore blockers.

In one aspect, compounds of the invention capable of modulating intracellular calcium levels inhibit electrophysiological currents directly associated with activation of SOC channels (I)_SOC). On the other hand, compounds capable of modulating intracellular calcium levels inhibit the electrophysiological currents directly associated with the activation of CRAC channels (I)_CRAC)。

The compounds of the invention are useful for treating diseases, conditions, or disorders that benefit from modulation of intracellular calcium, including, but not limited to, immune system-related diseases (e.g., autoimmune diseases), diseases or disorders involving inflammation (e.g., asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, uveitis, and immune system disorders), cancer or other proliferative diseases, liver diseases or disorders, and kidney diseases or disorders. In one embodiment, the compounds described herein are used as immunosuppressive agents to prevent (or inhibit) transplant rejection, allograft or xenograft rejection (organs, bone marrow, stem cells, other cells and tissues), and/or graft versus host disease. For example, the compounds of the present invention may be used to prevent (or inhibit) transplant rejection resulting from tissue or organ transplantation. The compounds of the invention may also be used to prevent (or inhibit) graft versus host disease caused by bone marrow or stem cell transplantation.

More particularly, compounds of formula I, IA-I, IA-II, IA-III and/or 1A-IV are useful for treating a variety of inflammatory diseases including, but not limited to, inflammation, glomerulonephritis, uveitis, liver disease or disorder, kidney disease or disorder, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, vasculitis, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allo-or xeno-transplantation, transplant rejection, graft-versus-host disease, lupus erythematosus, type I diabetes, pulmonary fibrosis, dermatomyositis, thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, hepatitis, and atopic dermatitis, Asthma and sjogren's syndrome.

The compounds described herein modulate the activity of, modulate the interaction with, or bind to or interact with at least a portion of a protein in the calcium pool-operated calcium channel complex. In one embodiment, the compounds described herein modulate the activity, modulate the interaction, or bind to or interact with at least a portion of the protein in a calcium release-activated calcium channel complex. In one embodiment, the compounds described herein reduce the level of functional calcium pool-manipulating calcium channel complexes. In another embodiment, the compounds described herein reduce the level of activated cisterna contributive calcium channel complexes. In yet another embodiment, the calcium pool manipulated calcium channel complex is a calcium release activated calcium channel complex.

A compound of the invention capable of modulating intracellular calcium levels for use in treating a disease or disorder is effective to reduce, ameliorate, or eliminate the symptoms or manifestations of the disease, condition, or disorder when administered to a subject having the disease or disorder. In other embodiments, a compound described herein is administered to a subject predisposed to a disease, condition, or disorder that does not yet exhibit symptoms of the disease, condition, or disorder, to prevent or delay development of the disease, condition, or disorder symptoms. In further embodiments, the compounds of the present invention have such an effect, either alone or in combination with other agents, or act to enhance the therapeutic effect of another agent.

Another embodiment of the invention is a method of treating a proliferative disease via modulation of calcium by administering to a patient in need of such treatment an effective amount of at least one compound of formula I, IA-I, IA-II, IA-III and/or IA-IV as defined above.

Yet another embodiment of the present invention is a method of treating a proliferative disease via calcium modulation by administering to a patient in need of such treatment an effective amount of at least one compound of formula I, IA-I, IA-II, IA-III and/or 1A-IV as defined above, in combination (simultaneously or sequentially) with at least one other anti-cancer agent. In one embodiment, the proliferative disease is cancer.

More particularly, compounds of formula I, IA-I, IA-II, IA-III, and/or IA-IV, and pharmaceutically acceptable esters or salts thereof, can be administered to treat, prevent, and/or ameliorate diseases or disorders in which calcium is implicated, including but not limited to cancer and other proliferative diseases or disorders.

Compounds of formula I, IA-I, IA-II, IA-III and/or IA-IV are useful for treating a variety of cancers, including but not limited to the following:

hematopoietic tumors of lymphoid lineage: leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma;

hematopoietic tumors of myeloid lineage: acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia;

cancer, including bladder, breast, colon, kidney, liver, lung, small cell lung, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin (including squamous cell carcinoma);

tumors of mesenchymal origin: fibrosarcoma and rhabdomyosarcoma;

tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas and schwannomas;

other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, follicular thyroid carcinoma and kaposi's sarcoma.

Since calcium is often a key role in regulating cell proliferation, calcium channel inhibitors may act as reversible cytostatics and may be useful in the treatment of any disease process characterized by abnormal cell proliferation, such as benign prostate hyperplasia, familial adenomatous polyposis, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplant rejection, endotoxic shock and fungal infections.

The compounds of the invention, as modulators of apoptosis, are useful in the treatment of cancer (including but not limited to those types mentioned herein above), viral infections (including but not limited to hepatitis virus, poxviruses, epstein-barr virus, sinders virus, and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus, lupus erythematosus, autoimmune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes), neurodegenerative diseases (including but not limited to alzheimer's disease, AIDS-related dementia, parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration), myelodysplastic syndrome, aplastic anemia, myocardial infarction-related ischemic injury, and the like, Stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol-related liver disease, hematological disorders (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteoporosis and arthritis), aspirin-sensitive sinusitis, cystic fibrosis, multiple sclerosis, renal disease, and cancer pain.

The compounds of the invention can modulate the level of cellular RNA and DNA synthesis. These agents are therefore useful for treating viral infections (including but not limited to HIV, human papilloma virus, poxviruses, epstein-barr virus, sinders virus and adenovirus).

The compounds of the invention are useful for chemoprevention of cancer. Chemoprevention is defined as the inhibition of invasive cancer or the inhibition of tumor recurrence by blocking the initiation of mutagenic events or blocking the progression of pre-cancerous cells that have been damaged. The compounds are also useful for inhibiting tumor angiogenesis and metastasis.

The compounds of the invention are also useful in combination (administered together or sequentially) with known anti-cancer therapies (e.g., radiotherapy) or with cytostatic or cytotoxic or anti-cancer agents, such as, but not limited to, DNA-interacting agents, e.g., cisplatin or doxorubicin; topoisomerase II inhibitors, such as etoposide; naturally occurring or synthetic topoisomerase I inhibitors, such as CPT-11 or topotecan; a tubulin interacting agent, such as paclitaxel, docetaxel, or an epothilone (e.g., ixabepilone); hormonal agents, such as tamoxifen; thymidylate synthase inhibitors, such as 5-fluorouracil; and antimetabolites (e.g., methotrexate), other tyrosine kinase inhibitors (e.g., Iressa and OSI-774); an angiogenesis inhibitor; an EGF inhibitor; a VEGF inhibitor; a CDK inhibitor; SRC inhibitors, c-Kit inhibitors; her1/2 inhibitors and monoclonal antibodies against growth factor receptors, such as Erbitux (EGF) and herceptin (Her2) and other protein kinase modulators.

The invention further provides pharmaceutical compositions comprising one or more compounds of formula I, IA-I, IA-II, IA-III and/or IA-IV and a pharmaceutically acceptable carrier.

Yet another embodiment of the invention is a dosage form comprising one or more compounds of the invention, optionally with a pharmaceutically acceptable carrier. The dosage form may be, for example, a solid oral dosage form, such as a tablet or capsule.

Drawings

For the present invention to be readily understood and put into practical effect, preferred embodiments will now be described, by way of example, with reference to the accompanying drawings, wherein like reference numerals designate like parts, and in which:

FIG. 1 is a photograph of a gel showing mRNA expression of Orai1 and STIM1 in A549 and NCI-H460 cell lines. Jurkat mRNA was used as a control.

Figure 2 is a graph of the percent inhibition of thapsigargin-induced calcium flux versus log of compound a concentration.

FIG. 3 is a graph of percent inhibition of NCI-H460 cell proliferation versus log concentration of compound A.

FIG. 4 is a photograph of a gel showing the effect of Compound B on Orai and STIM expression in the NCI-H460 cell line.

FIG. 5 is a graph of tumor volume in female Balb/c nude mice bearing NCI-H460 non-small cell lung cancer xenografts treated with vehicle, paclitaxel, or Compound A.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which the claimed subject matter belongs. Where there are multiple definitions of terms herein, the definitions in this section apply.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless otherwise indicated. Furthermore, the use of the term "including" as well as other forms is not limiting.

Definitions for standard chemical and MOLECULAR biological terms can be found in THE references, including but not limited to Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY, version 4", volumes A (2000) and B (2001), Plenum Press, New York and "MOLECULAR BIOLOGY OF THE CELL, version 5" (2007), Garland Science, New York. Unless otherwise indicated, mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacological routine methods are encompassed within the scope of the embodiments disclosed herein.

Unless specific definitions are provided, nomenclature employed in connection with the analytical and medicinal chemistry described herein, and the laboratory procedures and techniques thereof, are those commonly employed. In some embodiments, standard techniques are used for chemical analysis, drug preparation, formulation and delivery, and treatment of patients. In other embodiments, standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). In preferred embodiments, the reaction and purification techniques are performed, for example, using manufacturer's instructions or a kit as described herein. The foregoing techniques and steps have been generally performed by conventional methods and as described in various general and more specific references that are cited and discussed throughout this specification.

The following definitions as used herein shall apply unless otherwise indicated. Further, many of the groups defined herein may be optionally substituted. The list of substituents in the definitions is exemplary and is not to be construed as limiting the substituents defined elsewhere in the specification.

The term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, free of unsaturation, having from 1 to 8 carbon atoms and attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl and 1, 1-dimethylethyl (tert-butyl).

The term substituted or unsubstituted (C)_1-6) Alkyl refers to an alkyl group as defined above having up to 6 carbon atoms.

The term "alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched chain having from about 2 to about 10 carbon atoms, such as ethenyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl and 2-butenyl.

The term substituted or unsubstituted (C)_1-6) Alkenyl refers to an alkenyl group as defined above having up to 6 carbon atoms.

The term "alkynyl" refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and having from about 2 to 12 carbon atoms (presently preferred groups having from about 2 to 10 carbon atoms), such as ethynyl, propynyl, and butynyl.

The term substituted or unsubstituted (C)_1-6) Alkynyl refers to an alkynyl group as defined above having up to 6 carbon atoms.

The term "alkoxy" denotes an alkyl group as defined above attached to the rest of the molecule by an oxygen linkage. A typical example of those groups is-OCH₃and-OC₂H₅。

The term "cycloalkyl" denotes a non-aromatic mono-or polycyclic ring system of about 3 to 12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Non-limiting examples of polycyclic cycloalkyl groups include perhydronaphthyl, adamantyl, norbornyl (a bridged ring group), or spiro groups, such as spiro (4, 4) non-2-yl.

The term "cycloalkylalkyl" refers to a cyclic ring-containing group containing about 3 to 8 carbon atoms directly attached to an alkyl group, and then attached to the main structure at any carbon in the alkyl group that results in the creation of a stable structure, such as cyclopropylmethyl, cyclobutylethyl, and cyclopentylethyl.

The term "cycloalkenyl" refers to cyclic ring-containing groups containing from about 3 to 8 carbon atoms and at least one carbon-carbon double bond, such as cyclopropenyl, cyclobutenyl, and cyclopentenyl.

The term "aryl" refers to an aromatic group having 6 to 20 carbon atoms, such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl.

The term "arylalkyl" refers to an aryl group as defined above directly bonded to an alkyl group as defined above, e.g., -CH₂C₆H₅and-C₂H₅C₆H₅。

The term "heterocycle" refers to a non-aromatic 3-15 membered ring consisting of carbon atoms and at least one heteroatom selected from nitrogen, phosphorus, oxygen, and sulfur. For purposes of this invention, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, which can include fused, bridged, or spiro ring systems, and the nitrogen, phosphorus, carbon, oxygen, or sulfur atoms in the heterocyclyl group can be optionally oxidized to various oxidation states. In addition, the nitrogen atoms may optionally be quaternized. The heterocyclic group may be attached to the primary structure at any heteroatom or carbon atom that results in the creation of a stable structure.

The term "heteroaryl" refers to an optionally substituted 5-14 membered aromatic ring having as ring atoms one or more heteroatoms selected from N, O and S. Heteroaryl groups can be monocyclic, bicyclic, or tricyclic ring systems. Examples of such heteroaryl ring groups include, but are not limited to, oxazolyl, thiazolyl, imidazolyl, pyrrolyl, furyl, pyridyl, pyrimidinyl, pyrazinyl, benzofuryl, indolyl, benzothiazolyl, benzoxazolyl, carbazolyl, quinolinyl, and isoquinolinyl. The heteroaryl ring group may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.

Examples of such "heterocyclic" or "heteroaryl" groups include, but are not limited to, azetidinyl, acridinyl, benzodioxolyl, benzodioxanyl, benzofuranyl, carbazolyl, cinnolinyl, dioxolanyl, indolizinyl, naphthyridinyl, perhydroazepanyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pyridyl, pteridinyl, purinyl, quinazolinyl, quinoxalyl, quinolinyl, isoquinolinyl, tetrazolyl, imidazolyl, tetrahydroisoquinolinyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazaazapyrrolidinylZ is aza radicalExamples of the substituent include a substituent such as a phenyl group, a pyrrolyl group, a 4-piperidonyl group, a pyrrolidinyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, an oxazolyl group, an oxazolinyl group, an oxazolidinyl group, a triazolyl group, an indanyl group, an isoxazolyl group, an isoxazolidinyl group, a morpholinyl group, a thiazolyl group, a thiazolinyl group, an isothiazolyl group, a quinuclidinyl group, an isothiazolidinyl group, an indolyl group, an isoindolyl group, an indolinyl group, an isoindolyl group, an octahydroindolyl group, an octahydroisoindolyl group, a quinolyl group, an isoquinolyl group, a decahydroisoquinolyl group, a benzimidazolyl group, a thiadiazolyl group, a benzopyranyl group, a benzothiazolyl group, a furanyl group, a tetrahydrofuranyl group, a tetrahydropyranyl group, a thienyl group, a benzothienyl group, a thiomorpholinyl sulfoxide, a thiom.

The term "heteroarylalkyl" refers to a heteroaryl ring group as defined above bonded directly to an alkyl group. The heteroarylalkyl group may be attached to the main structure at any carbon atom from the alkyl group that results in the creation of a stable structure.

The term "heterocyclylalkyl" refers to a heterocyclyl group, as defined above, directly bonded to an alkyl group. The heterocyclylalkyl group may be attached to the primary structure at any carbon atom in the alkyl group that results in the creation of a stable structure.

Unless otherwise indicated, the term "substituted" means substituted with any one or any combination of the following substituents: hydrogen, hydroxy, halogen, carboxy, cyano, nitro, oxo (═ O), thio (═ S), substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted heterocyclylalkyl ring, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted guanidine, -COOR^x、-C(O)R^x、-C(S)R^x、-C(O)NR^xR^y、-C(O)ONR^xR^y、-NR^yR^z、-NR^xCONR^yR^z、-N(R^x)SOR^y、-N(R^x)SO₂R^y、-(＝N-N(R^x)R^y)、-NR^x C(O)OR^y、-NR^xR^y、-NR^xC(O)R^y-、-NR^xC(S)R^y-NR^xC(S)NR^yR^z、-SONR^xR^y-、-SO₂NR^xR^y-、-OR^x、-OR^xC(O)NR^yR^z、-OR^xC(O)OR^y-、-OC(O)R^x、-OC(O)NR^xR^y、-R^xNR^yC(O)R^z、-R^xOR^y、-R^xC(O)OR^y、-R^xC(O)NR^yR^z、-R^xC(O)R^x、-R^xOC(O)R^y、-SR^x、-SOR^x、-SO₂R^xand-ONO₂Wherein R in each of the above groups^x、R^yAnd R^zCan be a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted arylalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted heterocyclylalkyl ring, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted heterocycle, or R^x、R^yAnd R^zMay be linked to form a substituted or unsubstituted, saturated or unsaturated 3-10 membered ring, which may optionally comprise the same or different and is selected from O, NR^XOr a heteroatom of S. The substituents in the above "substituted" groups may not be further substituted. For example, when a substituent on a "substituted alkyl" is a "substituted aryl," a substituent on a "substituted aryl" may not be a "substituted alkenyl. Substituents or combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable compounds.

The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

The term "protecting group" or "PG" refers to a substituent that is used to block or protect a particular functionality. Other functional groups on the compound may remain reactive. For example, an "amino protecting group" is a substituent that is attached to an amino group, blocks or protects the amino functionality in a compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, t-Butyloxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Similarly, "hydroxy protecting group" refers to a hydroxy substituent that blocks or protects the hydroxyl functionality. Suitable hydroxy protecting groups include, but are not limited to, acetyl and silyl. "carboxy protecting group" refers to a carboxy substituent that blocks or protects the carboxy functionality. Suitable carboxyl protecting groups include, but are not limited to, -CH₂CH₂SO2Ph, cyanoethyl, 2- (trimethylsilyl) ethyl, 2- (trimethylsilyl) ethoxymethyl, 2-tosyl) ethyl, 2- (p-nitrophenylsulfinyl) ethyl, 2-diphenyl-l-phosphino) -ethyl and nitroethyl. General description of protecting Groups and their use is given in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley&Sons，New York，1991。

The term "stereoisomers" refers to compounds having the same chemical composition, but differing with respect to the arrangement of atoms and groups in space. These include enantiomers, diastereomers, geometric isomers, atropisomers or conformers.

All stereoisomers of the compounds described herein are within the scope of the invention. Racemic mixtures are also encompassed within the scope of the present invention. Thus, single stereochemical isomers and mixtures of enantiomers, diastereomers and geometric (or conformational) isomers of the compounds of the present invention are intended to be within the scope of the present invention.

The term "tautomer" refers to a compound characterized by the relative ease of interconversion of isomeric forms at equilibrium. These isomers are intended to be included in the present invention.

The term "prodrug" refers to an inactive precursor of a compound that is converted in vivo by normal metabolic processes to its active form.

The term "ester" refers to a compound formed by the removal of water by reaction between an acid and an alcohol. An ester can be represented by the formula RCOOR 'where R is a base compound and R' is an ester moiety (e.g., ethyl).

In addition, the present invention also includes compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the substitution of deuterium for hydrogen, and the like.

Pharmaceutically acceptable salts forming part of the invention include salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn and Mn; salts of organic bases such as N, N' -diacetylethylenediamine, glucosamine, triethylamine, choline, hydroxide, dicyclohexylamine, metformin, benzylamine, trialkylamine and thiamine; chiral bases, e.g. alkylanilines, glycinols andphenylglycinol; salts of natural amino acids, such as glycine, alanine, valine, leucine, isoleucine, norleucine, tyrosine, cystine, cysteine, methionine, proline, hydroxyproline, histidine, ornithine, lysine, arginine, and serine; quaternary ammonium salts of the compounds of the invention with alkyl halides or alkyl sulfates, e.g. MeI and (Me)₂SO₄(ii) a Unnatural amino acids, such as D isomers or substituted amino acids; guanidine or substituted guanidine, wherein the substituents are selected from nitro, amino, alkyl, alkenyl, alkynyl, ammonium or substituted ammonium salts and aluminum salts. Salts may include acid addition salts, sulfates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, citrates, fumarates, succinates, palmoates, methanesulfonates, benzoates, salicylates, benzenesulfonates, ascorbates, glycerophosphates and ketoglutarates, where appropriate.

The term "subject" or "patient" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (e.g., chimpanzees) and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents, such as rats, mice and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the methods and compositions provided herein, the mammal is a human.

The term "treating" as used herein includes alleviating, ameliorating, or preventing the underlying cause of the symptoms, inhibiting the disease, disorder, or condition, e.g., arresting the development of the disease, disorder, or condition, ameliorating the disease, disorder, or condition, causing regression of the disease, disorder, or condition, ameliorating the condition caused by the disease, disorder, or condition, or preventing and/or treating the symptoms that terminate the disease, disorder, or condition.

The term "target protein" as used herein refers to a protein or a portion of a protein that is capable of binding to or interacting with a compound described herein, e.g., a compound capable of modulating a STIM protein and/or an Orai protein. In certain embodiments, the target protein is a STIM protein. In other embodiments, the target protein is an Orai protein, and in still other embodiments, the compound targets STIM and an Orai protein.

The term "STIM protein" refers to any protein located in the endoplasmic reticulum or plasma membrane that stimulates an increase in calcium flux into cells via CRAC channels. (STIM refers to an interstitial interacting molecule.) the term "STIM protein" as used herein includes, but is not limited to, mammalian STIM-1, such as human and rodent (e.g., mouse) STIM-1, Drosophila melanogaster D-STIM, nematode C-STIM, Anopheles gambiae STIM, and mammalian STIM-2, such as human and rodent (e.g., mouse) STIM-2. As described herein, such proteins have been identified as being involved in, and/or providing pool-manipulated calcium entry or regulation thereof, cytoplasmic calcium buffering and/or calcium levels in the intracellular calcium pool (e.g., endoplasmic reticulum), calcium mobilization into, within, or outside of the intracellular calcium pool.

It will be appreciated that by "activating" is meant that the STIM protein up-regulates, stimulates, enhances or promotes calcium flow into cells via CRAC channels. It is expected that interference between STIM proteins and CRAC channels may occur through direct or indirect molecular interactions. Suitably, the STIM protein is a transmembrane protein associated with or adjacent to a CRAC channel.

STIM1 is known in the art as an essential component of CRAC channel activation. The present inventors have observed that STIM1 and STIM2 are expressed in certain NSCLC cell lines. Furthermore, CRACM1/Orai1 and CRACM3/Orai3 are overexpressed in certain NSCLC cell lines. While not wishing to be bound by any particular theory, CRAC and STIM proteins may cause activation of a proliferative pathway of NSCLC cells in the following manner: (i) excessive dysregulation of STIM in NSCLC cells causes inappropriate accumulation of the plasma membrane of STIM and (ii) on the plasma membrane, STIM activates CRAC (by direct or indirect interaction), thereby causing excessive calcium influx into the cell and initiating transcription, proliferation and invasion in NSCLC cells. Thus, inhibition of the CRAC channel or STIM pathway is an effective treatment for NSCLC.

As used herein, "Orai protein" includes Orai1 (SEQ ID NO: 1 as described in WO 07/081,804), Orai2 (SEQ ID NO: 2 as described in WO 07/081,804), or Orai3 (SEQ ID NO: 3 as described in WO 07/081,804). The Orai1 nucleic acid sequence corresponds to GenBank accession No. NM-032790, the Orai2 nucleic acid sequence corresponds to GenBank accession No. BC069270 and the Orai3 nucleic acid sequence corresponds to GenBank accession No. NM-152288. As used herein, Orai refers to any Orai gene, such as Orai1, Orai2, and Orai3 (see table I of WO 07/081,804). As described herein, such proteins have been identified as being involved in, and/or providing pool-manipulated calcium entry or regulation thereof, cytoplasmic calcium buffering and/or regulation of calcium levels in the intracellular calcium pool (e.g., endoplasmic reticulum) or movement of calcium into, out of, or within the intracellular calcium pool. In alternative embodiments, the Orai protein may be labeled with a tag molecule, such as, by way of example only, an enzyme fragment, a protein (e.g., c-myc or other tag protein or fragment thereof), an enzyme tag, a fluorescent tag, a fluorophore tag, a chromophore tag, a Rama activated tag, a chemiluminescent tag, a quantum dot tag, an antibody, a radioactive tag, or a combination thereof.

The term "fragment" or "derivative" when referring to a protein (e.g., STIM, Orai) refers to a protein or polypeptide that substantially retains the same biological function or activity as the native protein in at least one assay. For example, a fragment or derivative of a protein is preferably referred to as retaining at least about 50% of the activity of the native protein, at least 75% or at least about 95% of the activity of the native protein, as determined by calcium flux detection.

As used herein, "ameliorating" refers to amelioration of a disease or condition or at least partial relief of symptoms associated with a disease or condition. As used herein, amelioration of symptoms of a particular disease, disorder, or condition by administration of a particular compound or pharmaceutical composition refers to any reduction in severity, delay in onset, slowing of progression, or reduction in duration, either permanent or temporary, persistent, or transient, due to or associated with administration of the compound or composition.

The term "modulate" as used herein refers to interacting with a target protein, either directly or indirectly, to alter the activity of the target protein, including, by way of example only, inhibiting the activity of the target or limiting or reducing the activity of the target.

The term "modulator" as used herein refers to an agent that alters the activity of a target (e.g., a target protein). For example, in some embodiments, a modulator causes an increase or decrease in the magnitude of a certain activity of a target as compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, the modulator is an inhibitor that decreases the size of one or more activities of the target. In certain embodiments, the inhibitor completely prevents one or more activities of the target.

As used herein, "modulation" with respect to intracellular calcium refers to any alteration or regulation of intracellular calcium, including, but not limited to, alteration of calcium concentration in the cytoplasm and/or intracellular calcium storage organelles (e.g., the endoplasmic reticulum), or alterations in the kinetics of influx, efflux and intracellular calcium flow. In one aspect, modulation refers to decreasing.

The term "inhibition" or "inhibitor" of SOC channel activity or CRAC channel activity as used herein refers to inhibition of calcium pool-operated calcium channel activity or calcium release-activated calcium channel activity.

The term "acceptable" with respect to a formulation, composition or ingredient as used herein means having no permanent deleterious effect on the physical condition of the subject being treated.

The terms "pharmaceutically acceptable" molecular entities and compositions, when administered to a human, are physiologically tolerated and do not produce allergies or similar adverse reactions, such as gastric upset and dizziness. Preferably, the term "pharmaceutically acceptable" as used herein refers to approval by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "pharmaceutical composition" refers to a mixture of a compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.

The compounds and pharmaceutical compositions of the present invention may be administered by a variety of routes of administration, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

The term "effective amount" or "therapeutically effective amount" as used herein refers to a sufficient amount of an agent or compound administered to alleviate to some extent one or more of the symptoms of the disease or condition being treated. The result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, a "therapeutically effective amount" is the amount of a compound of the invention required to provide a clinically significant reduction in disease symptoms. In some embodiments, an appropriate "effective" amount in any individual case is determined using techniques such as dose escalation studies.

The term "enhance" as used herein refers to increasing or prolonging the efficacy or duration of a desired effect. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhancing" refers to increasing or prolonging the efficacy or duration of the effect of the other therapeutic agent on the system. The term "potentiating effective amount" as used herein refers to an amount sufficient to potentiate the effect of another therapeutic agent in the intended system.

The term "carrier" as used herein refers to a relatively non-toxic chemical compound or agent that facilitates incorporation of the compound into a cell or tissue.

The term "diluent" refers to a chemical compound used to dilute a compound of interest prior to delivery. In some embodiments, diluents are used to stabilize the compounds because they provide a more stable environment. Salts dissolved in buffers (also providing pH control or maintenance) are utilized as diluents, including but not limited to phosphate buffers.

By "cancer cell" is meant a cell from the lung, including pre-malignant, neoplastic, pre-malignant, tumorigenic, non-tumorigenic, and lung cancer stem cells.

As used herein, "intracellular calcium" refers to calcium that is located intracellularly, without specifying a particular cellular location. Conversely, "cytosol" or "cytoplasm" with respect to calcium refers to calcium located within the cytoplasm of the cell.

As used herein, the effect on intracellular calcium is any alteration of any aspect of intracellular calcium, including but not limited to alterations in the level and location of intracellular calcium and the movement of calcium into, out of, or within cells or intracellular calcium pools or organelles. For example, in some embodiments, the effect on intracellular calcium is a change in a property of calcium flow, e.g., a kinetic, sensitivity, rate, amplitude, and electrophysiological characteristic, or a movement occurring in a cell or a portion thereof. In some embodiments, the effect on intracellular calcium is an alteration of any intracellular calcium regulatory process, including calcium pool-operated calcium entry, cytosolic calcium buffering, and calcium levels in or movement into, out of, or in the intracellular calcium pool. Any of these aspects are detected in various ways, including, but not limited to, assessing calcium or other ion (especially cation) levels, movement of calcium or other ions (especially cations), fluctuations in calcium or other ion (especially cation) levels, kinetics of calcium or other ion (especially cation) flow, and/or transport of calcium or other ions (especially cations) through the membrane. A change is any such statistically significant change. Thus, for example, in some embodiments, if the intracellular calcium within the test cell and the control cell are said to be different, this difference is a statistically significant difference.

Modulation of intracellular calcium is any alteration or modulation of intracellular calcium, including, but not limited to, alteration of calcium concentration or level in the cytoplasm and/or intracellular calcium storage organelles (e.g., endoplasmic reticulum), alteration of movement of calcium into, out of, and within cells or intracellular calcium pools or organelles, alteration of intracellular calcium location, and alteration of the kinetics or other properties of calcium flow into, out of, and within cells. In some embodiments, intracellular calcium regulation involves altering or modulating (e.g., reducing or inhibiting) calcium pool-operated calcium entry in an intracellular calcium pool or organelle, cytosolic calcium buffering, calcium levels, or movement of calcium into, out of, or in the intracellular calcium pool or organelle and/or basal or resting cytosolic calcium levels. Modulation of intracellular calcium involves altering or modulating receptor-mediated ion (e.g., calcium) movement, second messenger-manipulated ion (e.g., calcium) movement, uptake or release of ions (e.g., calcium) into or out of the calcium and/or intracellular compartments of a cell including, for example, endosomes and lysosomes.

As used herein, "involved" in a relationship between a protein and intracellular calcium or an aspect of intracellular calcium regulation refers to the concomitant or related reduction, alteration, or elimination of one or more aspects of intracellular calcium or intracellular calcium regulation when expression or activity of the protein in the cell is reduced, altered, or eliminated. Such alteration or reduction in expression or activity occurs due to altered expression of the gene encoding the protein or by altering the protein level. Thus, a protein involved in an aspect of intracellular calcium (e.g., calcium pool-operated calcium entry) is a protein that provides or participates in intracellular calcium or an aspect of intracellular calcium regulation. For example, the protein that provides sink-manipulated calcium entry is a STIM protein and/or an Orai protein.

As used herein, a protein that is a component of a calcium channel is a protein that participates in the formation of a multi-protein complex of the channel.

As used herein, "cation entry" or "calcium entry" into a cell is directed to the entry of a cation (e.g., calcium) to an intracellular location (e.g., cytoplasm) or to the lumen of an intracellular organelle or storage site. Thus, in some embodiments, cation entry is, for example, movement of a cation from the extracellular medium or from an intracellular organelle or storage site to the cytoplasm, or movement of a cation from the cytoplasm or extracellular medium to an intracellular organelle or storage site. The movement of calcium from intracellular organelles or storage sites to the cytoplasm is also referred to as "calcium release" from the organelles or storage sites.

As used herein, "cellular response" refers to any cellular response caused by the movement of ions into or out of a cell or within a cell. In some embodiments, the cellular response is associated with any cellular activity that is at least partially dependent on an ion (e.g., calcium). Such activity optionally includes, for example, cell activation, gene expression, endocytosis, exocytosis, cell trafficking, and non-apoptotic cell death.

As used herein, "immune cell" includes cells of the immune system and cells that perform a function or activity in an immune response, such as, but not limited to, T cells, B cells, lymphocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, mast cells, plasma cells, leukocytes, antigen presenting cells, and natural killer cells.

As used herein, "cytokine" refers to a soluble small molecule protein secreted by a cell, in some embodiments, that alters the behavior or properties of the secreting cell or another cell. Cytokines bind to cytokine receptors and trigger behavior or properties within the cell, such as cell proliferation, death, or differentiation. Exemplary cytokines include, but are not limited to, interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1. alpha., IL-1. beta. and IL-1 RA), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), oncostatin M, erythropoietin, Leukemia Inhibitory Factor (LIF), interferons, B7.1 (also known as CD80), B7.2 (also known as B70, CD86), TNF family members (TNF-. alpha.), TNF-. beta., LT-. beta., CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail) and MIF.

"calcium reservoir-operated calcium entry" or "SOCE" refers to a mechanism that coordinates the release of calcium ions from the intracellular calcium reservoir with the flow of ions through the plasma membrane.

Cellular calcium homeostasis is the result of the sum of regulatory systems involved in controlling intracellular calcium levels and movement. Cellular calcium homeostasis is achieved, at least in part, by calcium binding or movement of calcium across the plasma membrane into and out of and within the cell, by movement of calcium across the membranes of intracellular organelles including, for example, the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, and endocytic organelles including endosomes and lysosomes.

Movement of calcium through the cell membrane is performed by specialized proteins. For example, calcium from the extracellular space enters the cells through various calcium channels and sodium/calcium exchangers and is actively squeezed out of the cells by calcium pumps and sodium/calcium exchangers. Calcium is also released from internal calcium pools via inositol triphosphate or ryanodine receptors and is likely to be taken up by these organelles by means of calcium pumps.

Calcium enters cells through any of several general classes of channels, including but not limited to voltage-operated calcium (VOC) channels, calcium pool-operated calcium (SOC) channelsAnd a sodium/calcium exchanger operated in reverse mode. VOC channels are activated by membrane depolarization and are found in excitable cells like nerves and muscles, and in most cases not excitable cells. Under some conditions, Ca²⁺Also by Na manipulated in reverse mode⁺--Ca²⁺The exchanger enters the cell.

Endocytosis provides another method for cells to take up calcium from the extracellular medium through endosomes. In addition, some cells, such as exocrine cells, release calcium by exocytosis.

In mammalian cells, cytosolic calcium concentrations are tightly regulated by quiescent levels, typically estimated at 0.1 μ M, while extracellular calcium concentrations are typically about 2 mM. This tight regulation promotes signal transduction into and into cells by transient calcium flux across the plasma membrane and intracellular organelle membranes. There are a number of intracellular calcium transport and buffering systems present in cells for shaping intracellular calcium signals and maintaining low resting cytoplasmic calcium concentrations. In resting cells, the main components involved in maintaining basal calcium levels are the calcium pump and the exudates in the endoplasmic reticulum and plasma membrane. Interference with resting cytosolic calcium levels effects the transmission of this signal and causes defects in many cellular processes. For example, cell proliferation involves calcium signal sequence elongation. Other cellular processes include, but are not limited to, secretion, signal transduction, and fertilization, involving calcium signaling.

Cell surface receptors that activate phospholipase C (PLC) produce cytosolic Ca from intracellular and extracellular sources²⁺A signal. [ Ca ]²⁺]_iThe initial transient increase in (intracellular calcium concentration) is due to Ca²⁺Release from the Endoplasmic Reticulum (ER) is caused by the PLC product, inositol-1, 4, 5-triphosphate (P) in the ER₃) Open IP₃Receptor triggering (Streb et al Nature, 306, 67-69, 1983). Persistent Ca across the plasma membrane at a later stage²⁺Entry into the specialized calcium pool-operated calcium (SOC) channels in the plasma membrane ensues (in the case of immune cells, SOC channels are calcium release-activated calcium (CRAC) channels). Ca cell-operated Ca²⁺Entry (SOCE) is the evacuation of Ca²⁺Pool self-activation of Ca in plasma membranes²⁺Channels to aid in replenishing the calcium reservoirThe process of (Putney, Cell Calcium, 7, 1-12, 1986; Parekh et al, Physiol. Rev.757-810; 2005). SOCE not only supplies Ca for replenishing the calcium pool²⁺And self-produces sustained Ca that controls essential functions such as gene expression, cellular metabolism, and exocytosis²⁺Signal (Parekh and Putney, physics. rev.85, 757-.

Activation of antigen or Fc receptor in lymphocytes and mast cells causes Ca²⁺Release from the intracellular calcium pool, which in turn leads to Ca²⁺Flow through CRAC channels in the plasma membrane. Intracellular Ca²⁺The subsequent increase in (b) activates calpain, a phosphatase that regulates the transcription factor NFAT. In resting cells, NFAT is phosphorylated and left in the cytoplasm, but when dephosphorylated by calpain, NFAT moves to the nucleus and activates different genetic programs depending on the stimulation conditions and cell type. In response to infection responses and transplant rejection, NFAT pairs with the transcription factor AP-1(Fos-Jun) in the nucleus of "effector" T cells, thereby transactivating the cytokine gene, genes that regulate T cell proliferation, and other genes that coordinate active immune responses (Rao et al, Annu Rev Immunol, 1997; 15: 707-47). In contrast, in T cells recognizing autoantigens, NFAT is activated in the absence of AP-1 and the transcription program inhibiting the autoimmune response, also known as "anergy", is activated (Macian et al, transcription mechanisms undersiding lymphocyte cancer. cell.2002, 6/14 th; 109 (6): 719-31). In a subset of T cells, termed regulatory T cells that suppress autoreactive effector T Cell-mediated autoimmunity, NFAT pairs with the transcription factor FOXP3 to activate genes responsible for suppressor function (Wu et al, Cell, 2006, 28.7.2006; 126 (2): 375-87; Rudensky A Y, Gavin M, Zheng Y.cell.2006, 28.7.2006; 126 (2): 253-.

The Endoplasmic Reticulum (ER) performs various processes. ER has Ca sensitivity as an agonist²⁺The role of the pool and the sink, protein folding/processing takes place in its lumen. At this time, Ca is abundant²⁺The dependent chaperones ensure that the newly synthesized protein is correctly folded and sent to the correct destination. ER is also involved in vesicle trafficking, stress signal release, cholesterolMetabolic regulation and apoptosis. Many of these procedures require intraluminal Ca²⁺And protein folding, ER stress response and apoptosis are likely all through Ca depletion²⁺ER for longer periods of time. Due to the Ca²⁺Source, apparently ER Ca²⁺The content must be reduced after stimulation. However, to preserve the functional integrity of ER, it is critical that Ca be present²⁺The content is not reduced too low or maintained at a low level. Thus supplementing ER with Ca²⁺Is a central process in all eukaryotic cells. Because of ER Ca²⁺Calcium pool-manipulated Ca in activated plasma membrane with reduced content²⁺Channels, so that the Ca²⁺The major function of the entry pathway is thought to be ER Ca, which is essential for maintaining proper protein synthesis and folding²⁺And (4) horizontal. However, Ca cell-operated Ca²⁺The channels have other important roles.

An understanding of the reservoir-operated calcium entry is provided by electrophysiological studies confirming that the process of emptying the reservoir activates what is known as Ca in mast cells²⁺Release of activated Ca²⁺Current or I_CRACThe current of (2). I is_CRACNon-voltage activated, commuted inward and for Ca²⁺Has obvious selectivity. The major hematopoietic origin is found in several cell types. I is_CRACNot only the reservoir-operated current, but it is now apparent that the reservoir-operated current encompasses Ca with different properties in different cell types²⁺A family of permeable channels. I is_CRACIs the expected first reservoir-operated Ca²⁺Current flow and is still a common mode of studying reservoir-manipulated flow.

The effect of a compound or agent on intracellular calcium may be monitored using various screening/identification methods that provide for direct or indirect assessment or measurement of the movement of calcium and/or ions of a cell (including cytosol and intracellular organelles or compartments) into, in or out of the cell, organelle, calcium pool, or part thereof (e.g., membrane). Calcium levels and ion movement or flux can be assessed using various methods. The particular method employed and the conditions employed will depend upon whether particular aspects of intracellular calcium are monitored or detected. For example, in some aspects, reagents and conditions can be used to specifically assess calcium pool-manipulated calcium entry, resting cytosolic calcium levels, calcium buffering and calcium levels, and calcium uptake or release by intracellular organelles and the calcium pool. Alternatively, the effect of a compound or agent on intracellular calcium can be monitored or detected using, for example, a cell, intracellular organelle or calcium reservoir, membrane (including, for example, split-membrane patches or lipid bilayers), or cell-free system (e.g., outer-facing outer membrane vesicles). Typically, some aspect of intracellular calcium is monitored or detected in the presence of a test agent and compared to a control, e.g., intracellular calcium in the absence of a test agent.

Diseases, disorders or conditions

Clinical studies have demonstrated that CRAC channels are absolutely required for gene activation in response to antigen by T cells. Lymphocyte activation and adaptive immune responses require sustained calcium entry. Calcium entry into lymphocytes occurs primarily through CRAC channels. Calcium elevation leads to the expression of cytokines required for NFAT activation and immune response. Inhibition of calcium pool-operated calcium entry is an effective way to prevent T cell activation.

Inhibition of CRAC channel activity with compounds that modulate intracellular calcium levels provides immunosuppressive therapy evidenced by the observation of pool-operated calcium entry elimination in Severe Combined Immunodeficiency (SCID) patients. T cells, fibroblasts and in some cases B cells from patients with T cell immunodeficiency or SCID, which have a major defect in T cell activation, show a strong defect in calcium pool-operated calcium entry. SCID patients lack adaptive immune response, but without any damage or toxicity in major organs. SCID patient phenotypes indicate that inhibition of CRAC channels is an effective strategy for immunosuppression.

Diseases/disorders involving inflammation and diseases/disorders related to the immune system

In some embodiments, the diseases, conditions, or disorders treated or prevented using the compounds capable of modulating intracellular calcium levels disclosed herein or compositions thereof and the methods provided herein of identifying compounds capable of modulating intracellular calcium levels include diseases, conditions, or disorders involving inflammation and/or associated with the immune system. These diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases (e.g., multiple sclerosis), and immune system disorders.

Activation of neutrophils (PMNs) by inflammatory mediators is achieved, in part, by increasing cytosolic calcium concentrations. It is believed that especially calcium pool-operated calcium flux plays an important role in PMN activation. Trauma has been demonstrated to increase PMN pool-manipulated calcium flow and prolonged elevation of cytosolic calcium concentrations due to enhanced pool-manipulated calcium flow is likely to alter coupling to stimuli responsive chemokines and to contribute to PMN dysfunction following injury. Thus, modulation of the concentration of PMN cytosolic calcium through calcium pool-operated calcium channels can be used to modulate PMN-mediated inflammation and spare cardiovascular function following injury, stroke or sepsis.

Calcium plays a key role in lymphocyte activation. For example, activation of lymphocytes by antigen stimulation causes a rapid increase in intracellular free calcium concentration and activation of transcription factors, including nuclear factor activating T cells (NFAT), NF- κ B, JNK1, MEF2, and CREB. NFAT is the major transcriptional regulator of the IL-2 (and other cytokines) gene. Maintenance of NFAT in a transcriptionally active state requires sustained elevation of intracellular calcium levels and is dependent on calcium pool-operated calcium entry. Reducing or blocking calcium pool-operated calcium entry in lymphocytes blocks calcium-dependent lymphocyte activation. Thus, in some embodiments, modulating STIM and/or Orai proteins, and in particular, calcium pool-manipulated calcium entry (e.g., reducing or eliminating calcium pool-manipulated calcium entry) in lymphocytes is a method of treating immune and immune-related disorders, including, for example, chronic immune diseases/disorders, acute immune diseases/disorders, autoimmune and immunodeficiency diseases/disorders, diseases/disorders involving inflammation, organ transplant rejection, and graft-versus-host disease, and alterations in immune responses (e.g., hyperactivity). For example, in some embodiments, treatment of an autoimmune disorder/condition involves reducing, blocking, or eliminating calcium pool-operated calcium entry in lymphocytes.

Examples of immune disorders include, for example, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, asthma, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allograft or xenograft (organ, bone marrow, stem cell and other cells and tissues) transplant rejection, graft versus host disease, lupus erythematosus, inflammatory disease, type I diabetes, pulmonary fibrosis, dermatomyositis, sjogren's syndrome, thyroiditis (e.g., hashimoto and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis, cystic fibrosis, chronic recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, and atopic dermatitis.

In other embodiments, the compounds capable of modulating intracellular calcium levels, compositions thereof, and methods provided herein for identifying compounds capable of modulating intracellular calcium levels are disclosed herein for use in conjunction with the treatment of malignancies, including but not limited to, malignancies of lymphoreticular endothelial origin, bladder cancer, breast cancer, colon cancer, endometrial cancer, head and neck cancer, lung cancer, melanoma, ovarian cancer, prostate cancer, and rectal cancer. Calcium pool-manipulated calcium entry is thought to play an important role in cancer cell proliferation.

SOCE inhibition is sufficient to prevent tumor cell proliferation. Pyrazole derivative BTP-2, a direct I_CRACA blocker that inhibits SOCE and proliferation in Jurkat cells and colon cancer cells. Furthermore, continuous SOCE requires mitochondrial Ca²⁺Uptake and prevention of mitochondrial Ca²⁺Uptake leads to SOCE inhibition. Stimulation of Jurkat cells to induce sustained SOCE and activate Ca to dephosphorylate NFAT²⁺The phosphatase-dependent calpain promotes the expression and proliferation of interleukin-2. In other embodiments, compounds capable of modulating intracellular calcium levels inhibit SOCE and are useful for treating cancer or other proliferative diseases or conditions.

Pool-operated calcium entry has been implicated in chronic liver disease and transplantation injury following cold storage-thermal deoxygenation.

In some embodiments, diseases, disorders, or conditions treated or prevented using the compounds capable of modulating intracellular calcium levels disclosed herein, compositions thereof, and methods provided herein to identify compounds capable of modulating intracellular calcium levels include renal or renal diseases and disorders. Mesangial cell proliferation is often a major feature of such diseases and conditions. In other embodiments, such diseases and conditions are caused by immunological or other damage mechanisms, including IgAN, membranoproliferative glomerulonephritis, or lupus nephritis. Imbalances in the control of mesangial cell replication also appear to play a key role in the pathogenesis of progressive renal failure. The turnover of mesangial cells in the kidney of normal adult is very low, with turnover rates below 1%. A significant feature of glomerular/renal disease is mesangial hyperplasia caused by an increased rate of mesangial cell proliferation or a decreased cell loss. Mesangial proliferative glomerulonephritis does not arise when proliferation of mesangial cells is induced without cell loss, for example, due to mitogenic stimulation. The data show that mesangial cell growth regulators, particularly growth factors, are thought to act by regulating calcium pool-manipulated calcium channels. In still other embodiments, the calcium pool-operated calcium flow modulator aids in the treatment of glomerular disease by inhibiting proliferation of mesangial cells.

In one aspect, the compounds described herein modulate intracellular calcium, such as, but not limited to, modulating (e.g., reducing or inhibiting) SOC channel activity, such as inhibiting CRAC channel activity (e.g., inhibiting I) in cells of the immune system (e.g., lymphocytes, leukocytes, T cells, B cells), fibroblasts (or fibroblast-derived cells), or epidermal, dermal or skin cells (e.g., keratinocytes)_CRACInhibit SOCE). In some embodiments, the step of modulating one or more proteins involved in modulating intracellular calcium (e.g., STIM proteins and/or Orai proteins) involves, for example, reducing the level, expression, activity, function, and/or molecular interaction of the proteins. For example, if a cell exhibits elevated calcium levels or lacks an aspect of intracellular calcium regulationSuch as calcium pool-operated calcium entry, in other embodiments, modulation involves reducing the level, expression, activity, or function of a protein (e.g., STIM protein and/or Orai protein) or molecular interaction.

Methods of identifying therapeutic agents for NSCLC

In one aspect, the invention provides a method of identifying a therapeutic agent for treating NSCLC, wherein the agent inhibits one or more plasma membrane calcium transport pathways. The methods include determining whether a candidate agent modulates all or part of the CRAC/STIM pathway in a NSCLC cell, thereby altering one or more cancer-associated properties of an epithelial cell.

By "cancer-associated property" is meant any physiological and/or pathological manifestation of a cell caused by a cellular cancer. Initiation of cell transcription, proliferation, cell death (e.g., apoptosis and necrosis), and invasion, including metastasis, migration, and loss of adhesion, are within the scope.

It will be appreciated that in a general format, candidate agents may directly modulate CRAC channels. In an alternative form, the candidate agent can modulate STIM protein, thereby altering calcium flux into the cancer cell.

In the context of the present invention, "altering" includes within its scope reducing, decreasing or down-regulating the calcium flow through the plasma membrane. It is contemplated that altering calcium flux into the cancer cell includes selectively altering calcium flux.

Easy to understand "regulate"," Modulator "or" Regulation"mechanisms" include within their scope any interaction that interferes with, inhibits, blocks or activates or increases the calcium flow-related activity of CRAC channels and/or STIM proteins. In certain embodiments, the modulator is an inhibitor. In other embodiments, the modulator is an antagonist. In yet other embodiments, the modulator is an agonist. In further embodiments, the modulator is an activator.

In one embodiment of the invention, the candidate agent selectively modulates a CRAC channel and/or STIM protein. In another embodiment, the candidate agent selectively inhibits CRAC channels and/or STIM proteins. In yet another embodiment, the candidate agent alters calcium flux by inhibiting CRAC channels and/or STIM proteins.

Thus, a modulator may be a peptide, protein (e.g., an antibody), or other organic molecule (e.g., a small organic molecule) with a desired biological activity and half-life. Both polyclonal and monoclonal antibodies directed against the entire protein or biologically active fragments thereof are contemplated as suitable modulators.

By "biologically active fragment" is meant a fragment, portion, region or segment of a protein that exhibits at least 10%, preferably at least 25%, more preferably at least 50% and more preferably at least 70%, 80% or 90% biological activity of the entire or full-length protein.

For CRAC channels, the biological activity is calcium transport activity. With respect to STIM proteins, biological activity is the ability to activate calcium transport into cells by interacting directly or indirectly with CRAC channels.

It will be appreciated by those of ordinary skill in the art that antibodies for human therapeutic applications must have specific properties that make these antibodies suitable for use in humans. Typically, a therapeutic antibody is "humanized", wherein the antibody typically comprises 90% human sequences and complementarity determining regions of a murine antibody. Humanized antibodies are particularly advantageous for medical applications due to the reduced likelihood of eliciting a foreign immune response.

It is contemplated that the humanized antibody may be directed against any STIM, such as, but not limited to STIM1 and STIM 2. In one embodiment, the humanized antibody is directed against STIM 1.

In other particular embodiments, the modulator is an antibody directed to a CRAC channel. In an alternative embodiment, the antibody directed to the CRAC channel is directed to CRACM 1.

Effective modulators are contemplated to include other potential CRAC channel inhibitors that may be useful according to the invention. Suitable examples include, but are not limited to, SKF-96365, T182, YM-58483, BTP-2, lanthanides (e.g., gadolinium), and other CRAC channel modulator compounds disclosed below, for example, PCT or U.S. patent applications assigned to Synta Pharmaceuticals, WO 2005/009954, WO 2005/009539, WO 2005/009954, WO 200/6034402, A1, WO 2006/081389, WO 2006/081391, WO 2007/087429, WO 2007/087427, WO 2007/087441, WO 2007/087442, WO 2007/087443, WO 2007/089904, WO 2007/109362, WO2007/112093, WO 2008/039520, WO 2008/063504, WO 2008/103310, WO 2009/017818, WO2009/017819, WO 2009/017831, US 2006/0173006 US 2007/0249051A 1, WO 2010/039238, WO 2007/109362, WO2007/112093, WO 2008/039520, WO 3982A 1, WO 2008/103310, WO 2009/017818, WO2009/017819, WO 2009/017831, US 2006/0173006, WO 2010/039237, WO2010/039236, WO2009/089305 and WO 2009/038775; astella, Queens Medical Center, Calcimedica and other patents and/or patent applications, including, i.e., WO 2007/121186, WO 2006/050214, WO 2007/139926, WO2008/148108, US 7,452,675, US 2009/023177, WO 2007/139926, US6,696,267, US6,348,480, WO2008/106731, US 2008/0293092, WO 2010/048559, WO 2010/027875, WO2010/025295, WO 2010/034011, WO2010/034003, WO 2009/076454, WO 2009/035818, US 2010/0152241, US 2010/0087415, US 2009/0311720 and WO 2004/078995.

Further suitable CRAC channel modulators include Isabella der et al Expert opinion in Drug Discovery 3(7) (2008), pp 787-800; young G et al, Cell Calcium 42(2007) 145-156; modulators disclosed in Yasurio Yonooky et al, Bio. & Med chem.14(2006) 4750-. All of these patents and/or patent applications and literature publications are incorporated herein by reference in their entirety for all purposes.

It is further contemplated that molecular biological methods for modulating the CRAC/STIM pathway may be employed. RNA interference, such as siRNA, provides a dramatic approach to silencing potential therapeutic gene targets by sequence-specific cleavage of homologous mrnas. Takeshita and Ochiya (Cancer Sci, 2006, 97: 689-.

The term "gene" is used herein to describe a discrete nucleic acid locus, unit and region within a genome that may contain one or more introns, exons, splice sites, open reading frames and 5 'and/or 3' non-coding regulatory sequences (e.g., promoters and/or polyadenylation sequences).

Thus, one skilled in the art will readily appreciate that the present invention contemplates a gene construct comprising one or more nucleotide sequences capable of directing the synthesis of an RNA molecule, wherein the nucleotide sequence is selected from the group consisting of:

(i) transcribable to comprise substantial homology to the RNA sequence encoded by the nucleotide sequence of interest

A nucleotide sequence of an RNA molecule of the RNA sequence;

(ii) (ii) the reverse complement of the nucleotide sequence in (i);

(iii) (iii) a combination of the nucleotide sequences in (i) and (ii);

(iv) (iv) multiple copies of the nucleotide sequence of (i), (ii) or (iii), optionally separated by a spacer sequence;

(v) (iii) a combination of the nucleotide sequences of (i) and (ii), wherein the nucleotide sequence of (ii) represents an inverted repeat of the nucleotide sequence of (i) separated by a spacer sequence; and

(vi) a combination as described in (v), wherein the spacer sequence comprises an intron sequence which is spliced from said combination.

If the nucleotide sequence comprises inverted repeats that are separated by non-intronic spacer sequences, once transcribed, the presence of the non-intronic spacer sequences facilitates the formation of stem-loop structures due to the association of the inverted repeats with each other. The presence of the non-intronic spacer sequence results in the formation of a transcribed RNA sequence (also referred to herein as a "transcript") in a form that can be referred to herein as a "hairpin" to maintain substantial integrity. Alternatively, if the nucleotide sequence comprises an inverted repeat in which the spacer sequence comprises an intron sequence, the intron/exon indirect sequences on either side of the intron sequence facilitate removal of sequences that will form loop structures once transcribed. The resulting transcript comprises a double-stranded rna (dsrna) molecule optionally having an overhanging 3' sequence at one or both ends. Such dsRNA transcripts are referred to herein as "perfect hairpins". An RNA molecule can comprise a single hairpin or multiple hairpins that include a single-stranded DNA "bulge" that occurs in a double-stranded DNA sequence.

Depending on the application, the RNA molecule may be directed against a single target or, alternatively, against several targets.

In certain embodiments, the RNA molecule encodes CRACM1/Orai1, CRACM2/Orai1 or CRACM3/Orai1 and/or STIM1 or STIM 2.

One skilled in the art will recognize that the therapeutic agents of the present invention can be identified by a number of methods for the treatment of cancer. Thus, the method of identifying a therapeutic agent includes determining whether a candidate agent can directly modulate a CRAC channel and/or modulate a STIM protein. In one embodiment, the method comprises determining whether a candidate agent can alter calcium flux into a cell by modulating CRAC channels and/or STIM proteins.

In one embodiment, a therapeutic agent of the invention for the treatment of NSCLC can be identified by screening a library of molecules, such as a library of synthetic chemicals (including combinatorial libraries), by methods such as those described in nester & Liu, 1998, comb.

It is also contemplated that naturally occurring libraries of molecules may be screened by known methods, for example, as described in Kolb, 1998, prog. Similarly, molecules can be identified by Molecular Library Programs (MLPs), such as those provided by the national health institute (NIH).

More rational approaches to designing therapeutics for treatment of NSCLC can employ more traditional biophysical techniques such as X-ray crystallography, NMR spectroscopy, computer-assisted screening of structural databases, computer-assisted modeling, or detection of molecular binding interactions as known in the art.

Structural bioinformatics can also be used to identify candidate agents for treating NSCLC. Fauman et al, 2003, meth. biochem. anal.44: 477 and Nature Reviews Drug Discovery 7, 783 (9 months 2008) present comments on the structural biological approach to Drug Discovery.

Computer-assisted structural database screening and bioinformatics approaches are increasingly being utilized for methods for identifying and/or engineering agonist and antagonist molecules. Examples of database screening methods can be found in U.S. Pat. No.5,752,019 and International publication No. WO 97/41526 (for identifying EPO mimetics) and U.S. Pat. Nos. 7,158,891 and 5,680,331 for more general computational methods for protein modeling and structural modeling of protein activity.

In general, other suitable methods include any of a variety of biophysical techniques for identifying molecular interactions. Such methods include, but are not limited to, competitive radioligand binding assays, electrophysiology, analytical ultracentrifugation, microcalorimetry, surface plasmon resonance, and methods based on optical biosensors, such as the method provided IN Coligan et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, 1997), Chapter 20, incorporated herein by reference.

One skilled in the art will appreciate that modulators may be in the form of binding partners and as such are identified by interaction assays such as yeast double-hybridization. A CURRENT protocolin PROTEIN SCIENCE (John Wiley & Sons, 1997), edited by Coligan et al, chapter 20, which is incorporated herein by reference, provides a two-hybrid screening method.

Pharmaceutical compositions and methods of treatment for NSCLC

It is also contemplated that, in one aspect, the invention provides a pharmaceutical composition comprising a therapeutic agent effective to treat a cancer identified by the above method and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the invention provides a method of treating cancer in a human by administering to the human a therapeutic agent effective to treat the cancer identified by the above method. Therapeutic agents, such as CRAC inhibitors, may be used as monotherapy or as adjunct therapy to one or more other methods of treatment of NSCLC.

In one embodiment, the therapeutic agent effective for treating cancer is in the form of a small organic molecule or peptide formulated in a pharmaceutically acceptable carrier, diluent or excipient suitable for oral administration, such as a transdermal patch or other non-invasive route of administration.

In yet another aspect, the invention includes a method of treating a patient suffering from NSCLC by administering to the patient an effective amount of a CRAC inhibitor. CRAC inhibitors mayAdjunctive therapy as monotherapy or as one or more other methods of treating lung cancer (or NSCLC), including chemotherapeutic agents for treating lung cancer, e.g. cisplatinEtoposide (VP-16;) CarboplatinPaclitaxelDocetaxelVinorelbine tartrateAdriamycinVincristine sulfateIsocyclophosphamide (ACS)And gemcitabine hydrochloride

As an adjunct therapy, CRAC inhibitors may be used with standard chemotherapy for lung cancer, typically exemplified by cisplatinEtoposide (VP-16;) CarboplatinPaclitaxelDocetaxelVinorelbine tartrateAdriamycinVincristine sulfateIsocyclophosphamide (ACS)And gemcitabine hydrochlorideA combination of two or more of (a). This standard chemotherapy combination therapy has been shown to enhance the overall response to treatment. Well-known drug pairings in standard chemotherapy combination therapy include paclitaxel + carboplatin, cisplatin + vinorelbine tartrate, cisplatin + etoposide, and carboplatin + etoposide. Current radiotherapy is often used with standard chemotherapy combinations of cisplatin + etoposide or carboplatin + etoposide.

Other chemotherapeutic agents useful for treating lung cancer include, for example, cyclophosphamideMethotrexate, lomustine (CCNU) and topotecan hydrochloride

For non-small cell lung cancer, asAdjuvant therapy, CRAC inhibitor and gemcitabine hydrochlorideA combination of chemotherapeutic agents that have unique activity against a number of solid tumors, including non-small cell lung cancer (NSCLC). Combination therapy with gemcitabine, cisplatin and vinorelbine tartrate has been found to be safe and very active in humans with advanced NSCLC. Another treatment option for NSCLC patients with advanced disease is alternating chemo-radiation therapy (e.g., cisplatin and etoposide followed by radiation therapy).

The term "pharmaceutically acceptable carrier, diluent or excipient" includes solid or liquid fillers, diluents or encapsulating substances that are safe for systemic administration. Depending on the particular route of administration, various carriers known in the art may be used. These carriers are selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline and salts, such as inorganic acid salts (including hydrochloride, bromate and sulfate salts) and organic acids (such as acetate, propionate and malonate salts) and pyrogen-free water.

Useful references describing pharmaceutically acceptable carriers, diluents and excipients are incorporated herein by reference, Remington's Pharmaceutical Sciences (Mack Publishing co.n.j.usa, 1991).

Any safe route of administration may be used to provide a patient with a therapeutic agent or pharmaceutical composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, and transdermal administration may be employed.

Suitable dosage forms include, but are not limited to, tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, and transdermal patches. These dosage forms may also include injection or implantation controlled release devices specifically designed for this purpose or other forms of implants modified to otherwise function in this manner. Controlled release of the therapeutic agent can be achieved by coating the therapeutic agent with, for example, hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release can be achieved using other polymer matrices, liposomes, and/or microspheres.

Pharmaceutical compositions of the invention suitable for oral or parenteral administration may be presented as discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any pharmaceutical method, for example, by bringing into association one or more agents described above with the carrier which constitutes one or more of the ingredients. The compositions can be prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then optionally shaping the product to give the desired appearance.

The above compositions can be administered in a manner compatible with the dosage form and in a pharmaceutically effective amount. In the context of the present invention, the dose administered to the patient should be sufficient to achieve a beneficial response in the patient after an appropriate period of time. The amount of agent administered will depend on the subject being treated (including age, sex, weight and general health thereof) and will depend on factors within the judgment of the physician.

Diagnostic method

In yet another embodiment, the invention is directed to a method of diagnosing NSCLC using the CRAC channel and STIM protein as diagnostic markers. In a particular aspect, the invention provides diagnostic methods for determining whether a patient is responsive to treatment with a therapeutic agent that alters calcium influx via the CRAC and/or STIM pathways by measuring the level of cancer cell endoplasmic membrane associated STIM.

In a particular embodiment, the present invention provides a diagnostic method for determining whether a human is susceptible to or suffering from NSCLC by detecting an excessive level of STIM protein in cancer cells, e.g., cells from the lung.

In another particular aspect, the diagnostic method of the invention comprises measuring the ratio of the STIM of one particular form to the STIM of another particular form.

In another embodiment, the invention provides diagnostic methods for detecting activation of CRAC channel expression in lung cells. It is envisioned that CRAC channel expression may be analyzed by protein-based or nucleic acid-based techniques.

"susceptible" and "susceptible" are used in the possible cases where an individual may exhibit clinical symptoms of NSCLC or any existing overt clinical symptoms of NSCLC are the underlying biochemical cause of the event.

One skilled in the art will readily appreciate that various methods can be used to measure the expression level of STIM on the plasma membrane of cancer cells. By way of example only, Fluorescence Activated Cell Sorting (FACS) analysis using labeled antibodies facilitates quantitative measurement of cell surface expression of proteins. For example, immunofluorescence and other fluorescence microscopy methods can also be used to stain tissue to detect STIM levels. Other conventional immunohistochemical techniques may also be used.

Alternatively, the relative protein expression levels may be determined by other protein-based methods, including immunodetection (e.g., ELISA and immunoblotting) to detect the relative expression levels of one or more proteins.

Proteomics profiling provides an alternative diagnostic method particularly for the analysis of the overall expression profile of proteins. Conrads et al, Expert Rev Mol Diagn.2003 July; 3(4): methods of cancer diagnosis using proteomic modalities are provided in 411-20 and incorporated herein by reference.

In particular embodiments, several proteins may be used in a protein library displayed in a variety of ways, such as phage display or cell display systems or by two-dimensional gel electrophoresis, or more particularly, differential two-dimensional gel electrophoresis (2D-DIGE). These particular embodiments may be generally referred to as "proteomics" or "proteomic profiling" methods, for example, as described IN CURRENT PROTOCOLS IN protencienc, ed by Coligan et al, ehhn Wiley & Sons NY USA (1996-2002), chapters 3.9.1 and 22.

In certain embodiments with respect to protein arrays, a cancer-associated protein of the invention (e.g., a NSCLC-associated protein) is located at an identifiable location on the array.

In exemplary embodiments, the protein array comprises a matrix immobilized, injected, bound or coupled to a cancer-associated protein (e.g., NSCLC-associated protein) or fragment thereof.

The substrate may be a chemically derivatized aluminum chip, a synthetic membrane, such as PVDF or nitrocellulose, a glass slide, or a microtiter plate.

Detection of the matrix binding protein can be performed using mass spectrometry, ELISA, immunohistochemistry, fluorescent microscopy, or by colorimetric detection.

The diagnostic methods of the invention may comprise measuring the expression level of a nucleic acid encoding a STIM protein and/or a CRAC channel. In this regard, nucleotide sequence variations in the promoter may, for example, affect the steady state levels of CRAC channel gene transcripts in one or more cells of an individual who is diseased or predisposed.

It is also contemplated that the relative levels of nucleic acids can be measured and/or compared in the diagnostic methods of the invention. For example, CRAC and/or STIM mRNA levels may be measured.

The use of a nucleic acid array can facilitate the relative level of a measured nucleic acid level compared to the expression level of a reference nucleic acid.

Nucleic acid array techniques have become well known IN the art and examples of methods that can be adapted for use IN array techniques are provided, for example, IN Current PROTOCOLS IN MOLECULAR BIOLOGY section 22 (John Wiley & Sons NY USA 1995 2001) compiled by Ausubel et al.

Arrays can be generated by a variety of methods, such as by photolithography (see, e.g., U.S. Pat. Nos. 5,143,854, 5,510,270, and 5,527,681), mechanical methods (e.g., directed flow methods as described in U.S. Pat. No.5,384,261), pin-based methods (e.g., as described in U.S. Pat. No.5,288,514), and magnetic bead technology (e.g., as described in International publication No. PCT/US 93/04145).

Reference is also made to the Affymetrix nucleic acid array system, such as described in U.S. Pat. Nos. 5,858,659 and 6,300,063, which provide a specific teaching of nucleic acid array-based detection of polymorphisms associated with disease.

In another particular form of this embodiment, quantitative or semi-quantitative PCR using primers corresponding to CRAC channel-encoding nucleic acids or STIM-encoding nucleic acids may be used to quantify the relative expression levels of CRAC channel nucleic acids or STIM nucleic acids, and thereby determine whether an individual is susceptible to or suffering from NSCLC.

PCR amplification is nonlinear, so end-point analysis does not always allow accurate determination of nucleic acid expression levels.

Real-time PCR analysis provides a high-throughput way to measure gene expression levels. It uses specific primers and fluorescence detection to measure the amount of product after each cycle. Hybridization probes utilize quenching dyes or fluorescence to directly generate a signal. This method is used to confirm and quantify differences in nucleic acid expression in cells or tissues obtained from cancer patients as compared to cells or tissues obtained from non-patients.

The following general methods described herein provide means and processes for making and using the compounds of the present invention and are intended to be illustrative, not limiting. Further modifications of the provided method and additional new methods can also be devised to achieve and serve the purpose of the present invention. It is therefore to be understood that other embodiments may exist which are within the spirit and scope of the invention as defined in the related specification.

General Process for the preparation of Compounds of formula (I)

The compounds of the present invention can be prepared by the following procedures. Unless otherwise indicated, when used in the following formulas, all variables will be understood to represent those groups described above with respect to formula (IA). These methods are similarly applicable to other compounds of formula (I) (e.g., (IA-I), (IA-II), (IA-III), and (IA-IV)).

Scheme 1 provides a general procedure for the synthesis of compounds of formula (IA), wherein L₁&L₂Together are-NH-CO-, R' "is hydrogen or halogen, and all other variables R, R¹、R²T, U, V, W, A and Cy are as described above for formula (IA).

The compound of formula 1 can be reacted with a compound of formula 2 (e.g., phenylhydrazine) to form a compound of formula 3. The concentrated H may then be used, for example₂SO₄And concentrated HNO₃Nitrating a compound of formula 3 to form a compound of formula 4. With FeCl in the presence of activated carbon₃And hydrazine reduction of the compound of formula 4 to produce the corresponding amine compound of formula 5a wherein R' "is hydrogen. Alternatively, halogenation after reduction of the compound of formula 4 yields the corresponding amine compound of formula 5b wherein R' "is halogen. In the presence of a suitable coupling agent, a compound of formula 5a or 5b may be coupled with various other intermediates to provide a compound of formula (IA). Compounds of formula 5a or 5b can be reacted with i.cy-a-COOH using one or more amide coupling agents, such as (benzotriazol-1-yloxy) tris (dimethylamino) phosphine hexafluorophosphate (BOP reagent) or N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC); an acid chloride of the formula Cy-A-COCl; or with an isocyanate of the formula Cy-NCO, wherein a is NH.

Scheme 2 provides a general procedure for the synthesis of compounds of formula (IA), wherein L₁&L₂Together are-NH-CO-, R' "is hydrogen or halogen, and all other variables R, R¹、R²T, U, V, W, A and Cy are as described above for formula (IA).

Step 1

Step 2

Step 1: the ketone of formula a can be condensed with the ester of formula b in the presence of a base, such as a metal alkoxide (e.g., sodium ethoxide) to produce the diketone of formula 1.

Step 2: the compound of formula 1 may be converted into the pyrazole compound of formula 2a by reacting the compound of formula 1 with hydrazine. In the presence of a suitable base, e.g. an alkaline metal carbonate (e.g. Cs)₂CO₃) There may be a compound of the formula 2a with L_gThe compound of formula 2b, which is a leaving group (e.g. halogen), is reacted to give a compound of formula 4, which compound of formula 4 may be subjected to a similar sequence of transformations as described above to give a compound of formula IA.

Scheme 2A provides a general procedure for the synthesis of compounds of formula (IA), wherein L₁&L₂Together are-CO-NH-, R' "is hydrogen or halogen, and all other variables R, R¹、R²T, U, V, W, A and Cy are as described above for formula (IA).

In the presence of a suitable base, e.g. an alkaline metal carbonate (e.g. Cs)₂CO₃) There may be a compound of the formula 2a with L_gThe compound of formula 2c, which is a leaving group (e.g., halogen), is reacted to produce a compound of formula 4a, and then the compound of formula 4a may be hydrolyzed to produce a compound of formula 5 c. The compound of formula 5c can be reacted with Cy-A-NH carbodiimide hydrochloride (EDC) using one or more amide coupling agents, such as (benzotriazol-1-yloxy) tris (dimethylamino) phosphine hexafluorophosphate (BOP reagent) or N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride₂And (3) coupling.

Analogous methods with certain modifications, as known to those skilled in the art, may be used to synthesize compounds of formula (I), (IA-I) or (IA-II) using suitable intermediates and reagents, wherein the variables are understood to represent those groups described above with respect to formula (I), (IA-II), (IA-III) or (IA-IV).

Experiment of

The following abbreviations are used throughout the disclosure: EDC.HCl [ N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride ]]HOBt [ hydroxybenzotriazole]TEA (triethylamine), DMF (dimethylformamide), AcOEt (ethyl acetate), DCM (dichloromethane), DMSO (dimethyl sulfoxide), THF (tetrahydrofuran). Unless otherwise mentioned, work-up (work-up) refers to the partitioning of the reaction mixture between aqueous and organic phases indicated in parentheses, separation and reaction over Na₂SO₄The organic layer was dried and the solvent was evaporated to obtain a residue. Unless otherwise stated in the context of the present invention,purification refers to column chromatography using silica gel as the stationary phase and a mixture of petroleum ether (boiling point 60-80 ℃) and ethyl acetate or dichloromethane and methanol of appropriate polarity as the mobile phase. RT (or RT) refers to ambient temperature (-25-28 ℃).

Intermediate 1: 1, 3-dicyclopropylpropane-1, 3-dione: sodium ethoxide (8g, 117.64mmol) was added to a solution of cyclopropylmethyl ketone (5g, 59.4mmol) and methyl cyclopropanecarboxylate (12mL, 118.9mmol) in DMSO (30 mL). The resulting mixture was heated at 60 ℃ overnight and then cooled to 0 ℃. After termination of the reaction with 6N HCl, work-up (H)₂O/AcOEt) yielded the title compound as a brown liquid, which was used without any purification.¹H-NMR(δppm，CDCl₃，400MHz)：16.05(bs，0.6H)，5.72(s，0.6H)3.78(s，0.8H)，2.08-2.0(m，0.8H)，1.62-1.53(m，1.2H)，1.12-1.05(m，4H)，0.97-0.83(m，4H).MS(m/z)：153.2[M+H]⁺。

Intermediate 2: 1-cyclopropyl-4, 4, 4-trifluorobutane-1, 3-dione: following a procedure similar to that described for intermediate 1. From cyclopropylmethyl ketone (10g, 119mmol), ethyl 2, 2, 2-trifluoroacetate (29mL, 237mmol), DMSO (60mL) and sodium ethoxide (16.1g, 237mmol) the title compound was obtained as a brown liquid (15g) which was used in the next step without purification.¹H-NMR(δppm，CDCl₃，400MHz)：5.65(s，2H)，2.16-2.04(m，1H)，1.18-1.12(m，2H)，0.98-0.94(m，2H)。

Intermediate 3: 3, 5-dicyclopropyl-1H-pyrazole: intermediate 1(5.3g, 35mmol) and hydrazine hydrate (1.8mL, 38.3mmol) in ethanol (20mL) were refluxed overnight. Cooling to ambient temperature post-treatment (H)₂O/AcOEt) yielded the title compound as a brown solid. M.P.: 161-164 ℃.¹H-NMR(δppm，CDCl₃，400MHz)：15.2(bs，1H)，5.65(s，1H)，2.16-2.09(m，2H)，1.18-1.14(m，4H)，0.98-0.94(m，4H).MS(m/z)：149.04[M+H]⁺。

Intermediate 4: 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazole: intermediate 2(0.120g, 0.66mmol) and hydrazine hydrate (0.04mL, 0.72mmol) were dissolved in ethanol (6mL) and refluxed overnight.After cooling the mixture to RT, work-up (H)₂O/AcOEt) gave the title compound (0.114g) as a brown solid.

Intermediate 5: 3, 5-dicyclopropyl-1- (4-nitrophenyl) -1H-pyrazole: intermediate 3(2.0g, 13.5mmol) and Cs were combined under nitrogen at 160 deg.C₂CO₃A solution of (5.51g, 40.5mmol) in DMSO (15mL) was heated for 0.5 h. 4-chloro-1-nitrobenzene (6.38g, 40.5mmol) was added to the mixture and stirred at the same temperature for 4 h. Treatment (H)₂O/AcOEt) and purification gave the title compound (0.8 g).¹H-NMR(δppm，CDCl₃，400MHz)：8.32(d，J 9.0，2H)，7.92(d，J 9.0，2H)，5.76(s，1H)，1.97-1.91(m，1H)，1.86-1.80(m，1H)，1.09-1.04(m，2H)，0.98-0.94(m，2H)，0.83-0.75(m，4H)。

Intermediate 6: 3, 5-dicyclopropyl-1- (2-fluoro-4-nitrophenyl) -1H-pyrazole: intermediate 3(2.0g, 13.5mmol) and K were reacted under nitrogen at 120 deg.C₂CO₃A solution of (5.5g, 40.6mmol) in DMSO (20mL) was heated for 0.5 h. To the mixture was added 3, 4-difluoro-1-nitrobenzene (2.15g, 13.5mmol) and stirred at the same temperature for 2 h. Treatment (H)₂O/AcOEt) and purification gave the title compound (3.16g) as a yellow solid.¹H-NMR(δppm，CDCl₃，400MHz)：8.19-8.12(m，2H)，7.78(t，J 7.9，1H)，5.70(s，1H)，2.10-2.00(m，1H)，1.68-1.58(m，1H)，1.08-0.92(m，4H)，0.82-0.74(m，2H)，0.72-0.65(m，2H)。

Intermediate 7: 2- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) -5-nitropyridine: intermediate 3(8.0g, 54.05mmol) and K were reacted under nitrogen at 110 deg.C₂CO₃A solution of (27.96g, 202.6mmol) in DMSO (60mL) was heated for 0.5 h. To the mixture was added 2-chloro-5-nitropyridine (12.8g, 80.75mmol) and stirred at the same temperature for 2 h. Treatment (H)₂O/AcOEt) and purification gave the title compound (3.03 g).¹H-NMR(δppm，CDCl₃，400MHz)：9.24(d，J 2.6，1H)，8.51(dd，J 2.6，9.9，1H)，8.10(d，J 9.2，1H)，5.72(s，1H)，2.90-2.75(m，1H)，1.99-1.90(m，1H)，1.06-0.93(m，4H)，0.82-0.64(m，4H)。

Intermediate 8: 5-cyclopropyl-1- (4-nitrophenyl) -3- (trifluoromethyl) -1H-pyrazole: a procedure similar to that followed for intermediate 5 was used. From intermediate 4(1.0g, 5.67mmol), Cs₂CO₃(5.5g, 16.9mmol), DMSO (4mL), and 4-chloro-1-nitrobenzene (1.93g, 14.1mmol) to obtain the title compound (0.7 g).¹H-NMR(δppm，CDCl₃，400MHz)：8.38(d，J 7.08，2H)，7.92(d，J 7.08，2H)，6.32(s，1H)，1.89-1.82(m，1H)，1.19-1.11(m，2H)，0.89-0.85(m，2H)，MS(m/s)：298.15[M+H]⁺。

Intermediate 9: 5-cyclopropyl-1- (2-fluoro-4-nitrophenyl) -3- (trifluoromethyl) -1H-pyrazole: intermediate 4(6.3g, 35mmol) and K were reacted under nitrogen at 120 deg.C₂CO₃A solution of (14.6g, 105mmol) in DMSO (20mL) was heated for 30 min. To the mixture was added 2-difluoronitrobenzene (5.68g, 35mmol) and stirred at the same temperature for 2 h. Treatment (H)₂O/AcOEt) and purification gave the title compound (7.52 g).¹H-NMR(δppm，DMSO-d₆，400MHz)：8.49(dd，J 2.4，9.9，1H)，8.47-8.27(m，1H)，8.04-8.02(m，1H)，6.73(s，1H)，1.76-1.68(m，1H)，0.99-0.90(m，2H)，0.84-0.74(m，2H)。

Intermediate 10: 2- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]-5-nitropyridine: intermediate 4(1.0g, 5.67mmol) and K were reacted at 90 ℃ under nitrogen₂CO₃A solution of (2.35g, 17.03mmol) in DMSO (10mL) was heated for 30 min. To the mixture was added 2-chloro-5-nitropyridine (1.35g, 8.5mmol) and stirred at the same temperature for 2 h. Treatment (H)₂O/AcOEt) and purification gave the title compound (0.30 g).¹H-NMR(δppm，CDCl₃，400MHz)：9.33(d，J 2.5，1H)，8.62(dd，J 2.8，9.0，1H)，8.19(d，J 9.0，1H)，6.29(s，1H)，2.92-2.83(m，1H)，1.60-1.50(m，2H)，0.79-0.70(m，2H)。

Intermediate 11: 2- [ 4-chloro-5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]-5-nitropyridine: intermediate 10(1.5g, 5.0mmol) was dissolved in DMF and N-chlorosuccinimide (0.8g, 6mmol) was added thereto at 0 ℃. The reaction was then allowed to stir at rt for 2 h. Completion of the reactionAfter this time, work up (EtOAc) and purification afforded the title compound (0.802 g).¹H-NMR(δppm，DMSO-d₆，400MHz)：9.34(d，J 2.5，1H)，8.65(dd，J 2.5，9，1H)，8.09(d，J 9，1H)，2.48-2.38(m，1H)，1.13-1.03(m，2H)，0.90-0.82(m，2H)。

Intermediate 12: 4- (3, 5-dicyclopropyl-1H-pyrazol-1-yl) aniline: towards EtOH/H₂A solution of intermediate 5(0.85g, 3.15mmol) in O (2: 1, 15mL) was added iron powder (0.88g, 15.8mmol) and ammonium chloride (17mg, 0.3mmol) and the mixture refluxed for 0.5 h. The mixture was filtered through celite and the celite was washed with ethanol. Concentration and merging layer post-treatment (H)₂O/AcOEt) to obtain the title compound (0.68g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：7.11(d，J 8.6，2H)，6.61(d，J 8.6，2H)，5.65(s，1H)，5.24(s，2H)，1.81-1.74(m，1H)，1.67-1.60(m，1H)，0.86-0.77(m，4H)，0.61-0.56(m，4H).MS(m/z)：240.3[M+H]⁺。

Intermediate 13: 4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) -3-fluoroaniline: intermediate 6(2g, 7.0mmol) in EtOH/H₂A solution in O (2: 1, 30mL) was added to iron powder (1.86g, 34.8mmol) and ammonium chloride (30mg, 0.7mmol) and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Treatment (H)₂O/AcOEt) and the combined layers were concentrated to give the title compound as a yellow solid (1.34 g).

Intermediate 14: 6- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) pyridin-3-amine: intermediate 7(0.77g, 2.86mmol) in EtOH/H₂A solution in O (2: 1, 15mL) was added to iron powder (0.79g, 14.17mmol) and ammonium chloride (15mg, 0.28mmol) and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Concentration and merging layer post-treatment (H)₂O/AcOEt) to yield intermediate 14(0.570g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：7.75(d，J 2.5，1H)，7.27(d，J 8.6，1H)，7.06(dd，J 2.7，8.6，1H)，5.67(s，1H)，5.43(s，2H)，2.39-2.27(m，1H)，1.88-1.74(m，1H)，0.90-0.72(m，4H)，0.69-0.50(m，4H)。

Intermediate 15: 4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]Aniline: following a procedure similar to that employed for intermediate 12. From intermediate 8(0.69g, 2.32mmol), EtOH-H₂O (2: 1, 12mL), Fe (0.64g, 15.8mmol) and NH₄Cl (0.012mg, 0.22mmol) to obtain the title compound (0.49g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：7.19(d，J 8.64，2H)，6.65(d，J 8.64，2H)，6.47(s，1H)，5.46(s，2H)，1.75-1.69(m，1H)，0.94-0.89(m，2H)，0.77-0.73(m，2H).MS(m/z)：268.1[M+H]⁺。

Intermediate 16: 4- [ 3-cyclopropyl-5- (trifluoromethyl) -1H-pyrazol-1-yl]-3-fluoroaniline: to intermediate 9(5g, 17.00mmol) in EtOH/H₂A solution in O (2: 1, 45mL) was added iron powder (4.75g, 85.1mmol) and ammonium chloride (90mg, 1.7mmol) and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Concentration and merging layer post-treatment (H)₂O/AcOEt) to obtain the title compound (4.3g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：7.16(t，J 8.5，1H)，6.50-6.45(m，3H)，5.86(s，2H)，1.60-1.51(m，1H)，0.91-0.82(m，2H)，0.76-0.69(m，2H)。

Intermediate 17: 6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]Pyridine-3-amine: to intermediate 10(0.77g, 2.86mmol) in EtOH/H₂A solution in O (2: 1, 9mL) was added iron powder (0.279g, 5.00mmol) and ammonium chloride (5mg, 0.09mmol) and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Concentration and merging layer post-treatment (H)₂O/AcOEt) to yield intermediate 17(0.239g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：7.84(d，J 2.6，1H)，7.33(d，J 8.6，1H)，7.12(dd，J 2.6，8.6，1H)，6.49(s，1H)，5.69(s，2H)，2.45-2.36(m，1H)，0.90-0.81(m，2H)，0.74-0.65(m，2H).MS(m/z)：269.2[M+H]⁺。

Intermediate 18: 6- [ 4-chloro-5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]Pyridine-3-amine: towards EtOH/H₂Intermediate in O (2: 1, 15mL)11(1.7g, 5.60mmol) iron powder (1.56g, 28.0mmol) and ammonium chloride (600mg, 11.2mmol) were added and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Treatment (H)₂O/AcOEt) and the combined layers were concentrated to give intermediate 18(1.1g) as a yellow solid.¹H-NMR(δppm，DMSO-d₆，400MHz)：8.04(s，1H)，7.39(d，J 8.2，1H)，7.20(d，J 8，1H)，4.26(s，2H)，2.10-1.99(m，1H)，1.96-1.85(m，2H)，1.84-1.70(m，2H)。

Intermediate 19: 2-chloro-N- [4- (3, 5-bicyclopropyl-1H-pyrazol-1-yl) phenyl]Acetamide: to a solution of intermediate 12(600mg, 2.24mmol) in Dichloromethane (DCM) was added chloroacetyl chloride (0.2mL, 2.39mmol) at 0 deg.C. The mixture was stirred for 15 min. Treatment (H)₂O/DCM) yielded intermediate 19, which was used in the next step without further purification.

Intermediate product 20: 2-chloro-N- {4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]Phenyl } acetamide: chloroacetyl chloride (0.05mL, 0.62mmol) was added to a solution of intermediate 15(150mg, 0.561mmol) in Dichloromethane (DCM) at 0 deg.C. The mixture was stirred for 15 min. Treatment (H)₂O/DCM) yielded the title compound, which was used in the next step without further purification.

Intermediate 21: 2-chloro-N- {6- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]Pyridin-3-yl } acetamide: to a solution of intermediate 17(500mg, 1.86mmol) in Dichloromethane (DCM) was added chloroacetyl chloride (0.16mL, 2.00mmol) at 0 deg.C. The mixture was stirred for 15 min. Treatment (H)₂O/DCM) yielded intermediate 21, which was used in the next step without further purification.

Intermediate 22: 5-cyclopropyl-1- (2-fluoro-4-iodophenyl) -3- (trifluoromethyl) -1H-pyrazole: to intermediate 16(1.9g, 7.20mmol) in 5ml of water was added concentrated HCl (5ml) and cooled to 0 ℃. To this solution was added nitrite solution (1g, 15mmol) slowly and stirred at 0 ℃ for 15 min. To this mixture was added potassium iodide solution (2.5g, 15mmol) at the same temperature and the reaction mixture was stirred at rt. Treatment (H)₂O/AcOEt) and purified to yield the desired product as a yellow liquid.¹H-NMR(δppm，DMSO-d₆，400MHz)：8.01(dd，J 1.7，9.5，1H)，7.79(dd，J 1.7，8.4，1H)，7.45(t，J 8.1，1H)，6.63(s，1H)，1.64-1.56(m，1H)，0.92-0.84(m，2H)，0.79-0.71(m，2H)。

Intermediate product 23: 4- [ 5-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-1-yl]-3-fluorobenzoic acid: magnesium (143mg, 6mmol) and a little iodine were suspended in diethyl ether under an inert gas. A small amount of methyl iodide was added thereto and the reaction mixture was refluxed to start grignard formation. Intermediate 22(790mg, 2mmol) was added at this stage and the reaction was continued under reflux. After complete consumption of the starting materials, the reaction mixture was cooled to rt and dry borneol was added thereto, followed by 2N HCl. The solid formed was filtered and dried under high vacuum to obtain the title compound as an off-white solid (160 mg).¹H-NMR(δppm，DMSO-d₆，400MHz)：13.6(bs，1H)，7.97-7.92(m，2H)，7.84-7.78(m，1H)，6.68(s，1H)，1.69-1.61(m，1H)，0.94-0.87(m，2H)，0.80-0.74(m，2H)。

Intermediate 24: 1H-benzo [ d ] imidazole-6-carboxylic acid: 3, 4-diaminobenzoic acid (5g, 32mmol) and formic acid (20ml) were mixed and refluxed overnight. Formic acid was removed with a rotary evaporator and water was added to the residue to obtain a solid. The solid was filtered and dried to obtain the title compound quantitatively.

Intermediate 25: 1H-benzo [ d ] [1, 2, 3] triazole-6-carboxylic acid: 3, 4-diaminobenzoic acid (5g, 32.8mmol) was dissolved in AcOH (30ml) and the mixture was cooled to 5 ℃. To this mixture was added NaNO2 solution (2.7g in 8ml water) followed by 2mi sulfuric acid. The reaction mixture was stirred for 90 min. After that, the reaction mixture was quenched with ice, and the obtained solid was filtered and washed with water to obtain the title compound (4.5g) as a brown solid.

Intermediate 26: 6-quinolinecarboxylic acid: to 4-aminophenylacetic acid (45g, 297mmol), glycerol (61.7g, 67mmol) and iodine (1.14g, 4mmol) was added dropwise sulfuric acid (67.5ml) at rt. The mixture was heated to 140 ℃ for 5 h. After this time, the reaction mixture was quenched with ice and the solid formed was filtered. The solid was dissolved in MeOH and charcoal was added to it and refluxed for 1 h. The mixture was filtered through celite and methanol was removed to obtain the title compound as a brown solid (7 g).

Intermediate 27: 6-Carboxylic acid quinoxaline: to an aqueous potassium carbonate solution (7ml, 1.82g K)₂CO₃) To this was slowly added 3, 4-diaminobenzoic acid (500mg, 3.29 mmol). Then, glyoxal bis (sodium sulfite) hydrate adduct (963mg, 3.62mmol) was slowly added. The mixture was heated to 80 ℃ for 5h to obtain a clear solution. After completion of the reaction, the reaction mixture was slowly added to dilute HCl, filtered and the solid formed was dried to obtain the title compound (300mg) as a brown solid.

Intermediate 28: 2- (imidazo [1, 2-a ]]Pyridin-2-yl) acetic acid ethyl ester: 2-aminopyridine (500mg, 5.31mmol) and ethyl chloroacetoacetate (870mg, 5.31mmol) were dissolved in DMSO and heated to 100 ℃ under inert gas for 1 h. After 1H, water is added to the reaction mixture, which is then worked up (H)₂O/AcOEt) and purified on 60-120 mesh silica using AcOEt and petroleum ether (30: 70) to give the title compound as a brown liquid (110 mg).¹H-NMR(δppm，CDCl₃，400MHz)：8.06(d，J 6.7，1H)，7.59(s，1H)，7.55(d，J 9.4，1H)，7.14(t，J 7.9，1H)，6.75(t，J 6.7，1H)，4.12(q，J 7.1，2H)，3.87(s，2H)，1.3(t，J 7.1，3H)。

Intermediate 29: 2- (imidazo [1, 2-a ]]Pyridin-2-yl) acetic acid: intermediate 28(7.5g, 39.22mmol) was dissolved in water (30ml) and NaOH (2.35g, 58.8mmol) was added. The mixture was heated to 90 ℃ for 1 h. Thereafter, water was removed by distillation and the reaction mixture was acidified to pH 7 with dilute HCl to obtain a solid. The solid was filtered and dried under vacuum to quantitatively obtain the title compound as a brown solid.¹H-NMR(δppm，CDCl₃，400MHz)：8.50(d，J 6.7，1H)，7.82(s，1H)，7.46(d，J 9，1H)，7.20(t，J 7.5，1H)，6.85(t，J 6.7，1H)，3.69(s，2H)。

Intermediate product 30: 2- (quinolin-6-yl) acetic acid: to 4-aminophenylacetic acid (45g, 297mmol), glycerol (61.7g, 67mmol) and iodine (1.14g, 4mmol) was added dropwise sulfuric acid (67.5 ml). The mixture was heated to 140 ℃ for 24 h. After that, the reaction mixture was cooled to rt and the pH was adjusted to 5 with 10% sodium hydroxide solution. To the direction ofTo this were added methanol (350ml) and sulphuric acid (3ml) and heated to 100 ℃ for 24 h. The reaction mixture was filtered through celite and the filtrate was evaporated with a rotary evaporator to obtain a residue. The pH of the residue was adjusted to 5 using 4% NaOH solution and extracted with EtOAc. With anhydrous Na₂SO₄The EtOAc layer was dried and the EtOAc was removed using a rotary evaporator to obtain the crude product. The crude product was purified by column using EtOAc and petroleum ether as eluent to obtain methyl 2- (quinolin-6-yl) acetate (11.2 g). Methyl 2- (quinolin-6-yl) acetate (11.2g) was dissolved in methanol (8ml) and water (8ml) and sodium hydroxide (3.3g, 82mmol) was added. The mixture was stirred for 30min and methanol was removed with a rotary evaporator to obtain a residue. The residue was acidified to pH 5 using 0.8N HCI to obtain a solid. The solid was filtered and dried to obtain the title compound (8.2 g).

Intermediate 31: 2- (3-nitropyridin-2-ylamino) acetic acid: 2-chloro-3-nitropyridine (5g, 31.Smmol) was dissolved in EtOH (125ml), potassium carbonate (4.35g, 31.5mmol) was added, and glycine (4.73g, 6.3mmol) in 25ml water was added to the mixture and refluxed overnight. The reaction mixture was cooled to 0 ℃ to obtain a solid. EtOH was then removed with a rotary evaporator and acidified with 2N HCl, the solid was filtered through a column and dried to quantitatively obtain the title compound as a yellow solid.

Intermediate 32: 2- (3-aminopyridin-2-ylamino) acetic acid: to intermediate 31(10g, 50.74mm0l) in EtOH/H₂A solution in O (2: 1, 225mL) was added iron powder (14.15g, 0.25mol) and ammonium chloride (5.41g, 101.47mmol) and the mixture was refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Treatment (H)₂O/AcOEt) and the combined layers were concentrated to give the title compound as a brown solid (10 g).

Intermediate 33: 2- (3H- [1, 2, 3] triazolo [4, 5-b ] pyridin-3-yl) acetic acid: intermediate 32(10.92g, 65.35mmol) was dissolved in 30ml water and 13ml AcOH was added. To this mixture was added a solution of sodium nitrite (4.96g, 71.88mmol) at rt and the mixture was cooled to 0 ℃. The reaction mixture was stirred for 30min and filtered. The obtained solid was dried under vacuum to obtain the title compound (7.5g) as a red solid.

Intermediate 34: (R) -2- (3-nitropyridin-2-ylamino) propionic acid: 2-chloro-3-nitropyridine (500mg, 3.15mmol) was dissolved in EtOH (12.5ml), potassium carbonate (435mg, 3.15mmol) was added and to the mixture was added (S) -2-aminopropionic acid (561mg, 6.3mmol) in 2.5ml water and refluxed overnight. The reaction mixture was cooled to 0 ℃ to obtain a solid. EtOH was then removed on a rotary evaporator and acidified with 2N HCl, the solid filtered and dried in vacuo to give the title compound as a yellow solid (460 mg).

Intermediate 35: (R) -2- (3-aminopyridin-2-ylamino) propionic acid: iron powder (657mg, 11.78mmol) and ammonium chloride (251mg, 53.4mmol) were added to EtOH/H₂Intermediate 34(500mg, 2.35mmol) in O (2: 1, 12mL) and the mixture refluxed for 1 h. The mixture was filtered through celite and the celite was washed with ethanol. Treatment (H)₂O/AcOEt) and the combined layers were concentrated to give the title compound as a black solid (600 mg).

Intermediate 36: (R) -2- (3H- [1, 2, 3)]Triazolo [4, 5-b]Pyridin-3-yl) propionic acid: intermediate 35(600mg, 3.3mmol) was dissolved in 1.5ml of water and 0.5mi AcOH was added. To the mixture was added a solution of sodium nitrite ((190mg, 2.76mmol) at rt and the mixture was cooled to 0 deg.C. the reaction mixture was stirred for 30min, then filtered.¹H-NMR(δppm，DMSO-d₆，400MHz)：11.04(s，1H)，8.18-8.17(m，1H)，7.51-7.40(m，2H)，5.30(q，J 7.12，1H)，1.18(d，J7.12，3H)。

Intermediate 37: 2- (quinolin-6-yl) propionic acid methyl ester: THF (5ml) was placed in the RBF, diisopropylamine (0.19ml, 1.29mmol) was added and cooled to-78 ℃ under nitrogen. N-butyllithium (0.8ml, 1.29mmol) was then added and stirred at the same temperature for 30 min. Methyl 2- (quinolin-6-yl) acetate (0.2g, 0.99mmol) was added at this stage and stirred at-78 ℃ for 30 min. Methyl iodide (0.17g, 1.2mmol) was then added and stirred at-78 ℃ for 30min, then slowly raised to rt. The reaction mixture was stirred at rt overnight. The reaction mixture was quenched with water and extracted with EtOAc. Through anhydrous Na₂SO₄Drying of organic matterThe layers were combined and EtOAc removed using a rotary evaporator to afford the crude product which was purified by column chromatography over 60-120 mesh silica gel using EA and petroleum ether (25: 75) as eluent.¹H-NMR(δppm，CDCl₃，400MHz)：8.89-8.86(m，IH)，8.13(d，J 7.8，1H)，8.07(d，J 8.7，1H)，7.76-7.64(m，2H)，7.42-7.37(m，1H)，3.92(q，J 7.2，1H)，3.68(s，3H)，1.60(d，J 7.2，3H)。

Intermediate 38: 2- (quinolin-6-yl) propionic acid: intermediate 37(440mg, 2.04mmol) was dissolved in MeOH (5ml) and water (2ml) and lithium hydroxide (427mg, 10.2mmol) were added. The mixture was refluxed for 2h and the reaction mixture was cooled. Methanol was removed with a rotary evaporator and 6N HCl was added to the residue to adjust the pH to 7. The obtained solid was filtered and dried to obtain the title compound as a solid.

Intermediate 39: quinolin-6-ylmethylamine: 6-cyanoquinoline (14g) [ synthesized according to Srivastava, Rajiv et al Synthetic Communications 37(3), 431-. After completion of the reaction, the reaction mixture was filtered through celite, and the celite bed was washed with MeOH. The filtrate was concentrated to give the title compound (13.5g) as a black syrup liquid.

General procedure for amide formation

Process 1: a solution of the appropriate aniline (1 eq), the essential acid (1.1 eq), edc.hcl (1.2 eq), HOBt (0.5 eq) and TEA (3 eq) in DMF was stirred at RT. Treatment (H)₂O/AcOEt) and purified to yield the desired product.

And (2) a process: the acid (1 eq) was dissolved in DCM, cooled to 0 ℃ and oxalyl chloride (3 eq) and 3 drops of DMF were added. The reaction mixture was stirred at room temperature for 30min and DCM was removed with a rotary evaporator to obtain the acid chloride. In N₂The amine was dissolved in DCM under gas and pyridine (1.3 eq) was added. To this mixture was added the acid chloride in DMC and allowed to stir at room temperature until the amine was completely consumed. Treatment (H)₂O/AcOEt) and purified to yield the desired product.

The following compounds were prepared using these procedures:

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