CN110114353A - Benzo [b] thiophene-carboxamides analog derivative and application thereof - Google Patents

Benzo [b] thiophene-carboxamides analog derivative and application thereof Download PDF

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CN110114353A
CN110114353A CN201780080343.5A CN201780080343A CN110114353A CN 110114353 A CN110114353 A CN 110114353A CN 201780080343 A CN201780080343 A CN 201780080343A CN 110114353 A CN110114353 A CN 110114353A
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cancer
alkyl
carbonyl
independently selected
aryl
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CN110114353B (en
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宋淳
张成城
黄牛
张承智
赵昕
张衡
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Jinan Chengcheng Biotechnology Co ltd
Shandong University
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Shandong University
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    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

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Abstract

Disclose benzo [b] thiophene-carboxamides analog derivative and application thereof.In particular it relates to the purposes of benzo [b] thiophene-carboxamides analog derivative or its pharmaceutically acceptable salt and these compounds in adjusting Smoothened receptor or Hedgehog pathway activity with formula (I) structure.

Description

Benzo [b] thiophene-carboxamides analog derivative and application thereof Technical field
The disclosure belongs to technical field of pharmaceuticals, specifically, it is related to benzo [b] thiophene-carboxamides analog derivative and its pharmaceutically acceptable salt and these compounds in adjusting Smoothened receptor or Hedgehog pathway activity or for treating the purposes in disease relevant to Smoothened receptor active or Hedgehog pathway activity.
Background technique
Hedgehog signal path (Hedgehog/Hh access) plays an important role in the histoorgan forming process of vertebrate early development, is responsible for adjusting cell Proliferation and differentiation.During vertebrate animal development, Hh access participates in control polarity, body segment and limbs left-right asymmetry, in CNS, organ occurs, cartilage occurs and the maturation of gonad.In adult tissue, on the one hand Hh signal path carries out fine feedback regulation to own signal access, the multiple functions behavior of another aspect regulating cell is such as: cell Proliferation, anti-apoptotic, vascularization, induction and maintenance stem cell properties, cell fate determine, epithelium mesenchyma converts, migration etc..Hh signal path includes four Major Members: Hh ligand, 12 transmembrane protein receptor Patched (Ptch), the transcription factor of seven-transmembrane protein receptor Smoothened (Smo) and Gli family.In vertebrate, Hh ligand mainly includes 3 hypotypes, Sonic hedgehog (Shh), Desert hedgehog (Dhh) and Indian hedgehog (Ihh).Ptch is 12 transmembrane proteins, and there are two homologous genes: Ptch1 and Ptch2.Seven-transmembrane Protein S mo is an important member in Hh signal path, belongs to Frizzled family, is one of GPCR superfamily.Gli is the transcription factor of Hh signal path most downstream, there is 3 homologous genes: Gli1, Gli2, Gli3 in vertebrate.Gli1 is an activating transcription factor, and Gli2 and Gli3 are difunctional transcription factors, and overall length form is activating transcription factor, and clipped form after being modified by proteasome Partial digestion is transcription inhibitory factor.In the presence of not having Hh ligand, Ptch inhibits downstream Smo protein active, and Sufu forms compound in conjunction with Gli, which forms the inhibition form (Gli of Gli through the cracking of Perhydrolase R), inhibit the activity of access.In the presence of Hh ligand, Ptch releases the inhibition of Smo, and the transcription factor Gli in Smo activation downstream enters nucleus, promotes the transcriptional expression of different target genes, realizes the activation of Hh signal path.Smo receptor conducts receptor as transmembrane signal crucial in Hh signal path, plays an important role during Hh signal transduction.
In adult organism, only least a portion of Hh signal path is active, and the stable state for tissue maintains and injury repair process, and major part is all silenced closing and falls.The Hh signal path of this part activation is able to maintain that the distribution of stem cell, regeneration and differentiation.Therefore, Smo small-molecule modulators can promote the differentiation of stem cell to repair the damage as caused by disease, such as promote revascularization, and treatment is damaged as caused by ischaemic；The proliferation and differentiation of osteogenic cell are adjusted, osteoporosis and osteopathy are treated；Parkinsonism and diabetic neuropathy are treated in the differentiation of adjusting maincenter and peripheral neurons cell.
But Hh signal path excessive activation, then take part in the generation of some malignant tumours and cancer.It is counted according to current AUTHORITATIVE DATA, there is the abnormal activation of Hh signal in the cancer of a quarter.The relevant cancer with Hh signal path abnormal activation activates the difference of form to be divided into three types according to Hh signal path in cancer: I type caused by being mutated, such as nevoid basal cell carcinoma syn drome, basal-cell carcinoma, pith mother cells cancer, rhabdomyosarcoma；The II type that ligand autocrine relies on, such as Small Cell Lung Cancer, cancer of pancreas, the cancer of the esophagus, gastric cancer, cholangiocarcinoma, colon cancer, prostate cancer, breast cancer, melanoma；The type III that ligand paracrine relies on, such as prostate cancer, cancer of pancreas, lymthoma, Huppert's disease, leukaemia.
Hh signal pathway inhibitor is in research and development and rises rapidly the stage for treating kinds of tumors disease at present.Along on January 30th, 2012, FDA had approved the small molecule Smo inhibitor Vismodegib of Genentech (Roche), was unsuitable for surgical advanced stage basal-cell carcinoma for treating.And the Novartis small molecule Smo inhibitor Sonidegib then ratified, demonstrate feasibility of the Smo as Hh pathway inhibitor target spot.Currently, having multiple small molecule Smo inhibitor medicaments to be in different development phases in the Hh inhibitor research and development of each major company, involved clinical indication is also many kinds of.Wherein the BMS-833923 of LY-2940680, BMS company exploitation of Erismodegib, Lilly company of Novartis company exploitation is in progress more smoothly in the clinical II phase, in addition, the second generation Smo inhibitor LEQ-506 of Novartis company is in clinical I phase research.Although having reported various inhibitors, the huge pharmaceutical value of Smo inhibitor is not fairly well-developed, and only indication is more narrow at present, and there are many drug resistance problems to report.In addition, the research of Hh agonist is gradually taken seriously, there are multiple compounds carrying out preclinical study.
Summary of the invention
In one aspect, the disclosure provides formula (I) described compound or its pharmaceutically acceptable salt,
Wherein:
R 1Selected from C 5-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；
R 2Selected from-H and-CH 3；
R 3Selected from-H, hydroxyl, alkoxy, C 1-C 10Alkoxy carbonyl group, C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 9aR 9b、-(CH 2) nOR 9a、-(CH 2) nCONR 9aR 9b、-CO-((CH 2) n-A) m1-((CH 2) n-B) m2-R 9a、-CO-(A-(CH 2) n) m-B-R 9a, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl are optionally further by one or more R 10Replaced group；
R 4、R 5、R 6It is each independently selected from-H ,-F ,-Cl ,-Br and-I, and R 5And R 6It cannot simultaneously be-H；
Work as R 7In the presence of, each R 7Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, C 1-C 10Alkyl, alkoxy, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 8aR 8b、-(CH 2) nOR 8a、-(CH 2) nCONR 8aR 8b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl are optionally further selected from replaced the groups of halogen, hydroxyl, amino, cyano, nitro and aldehyde radical by one or more；
R 8a, R 8b, R 9aAnd R 9bIt is each independently selected from hydrogen, C 1-C 10Alkyl, halogenated C 1-C 10Alkyl, C 1-C 10Alkyl amino, C 3-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl and C 6-C 10Aryl；Or R 8a/R 8bOr R 9a/R 9bThe N being connected with them is formed together 3 to 8 unit monocycles, and 3 to 8 unit monocycle is saturation or unsaturated, including with R 8a/R 8bOr R 9a/R 9bIncluding the nitrogen-atoms connected, the hetero atom of O, S or N are each independently selected from 3 to 8 unit monocycles containing one or more；
R 10Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy, C 6-C 10Aryl and-NR 8aR 8b；
A and B are each independently selected from-O- and-NH-；
m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6；
N is 0,1 or 2.
In some embodiments, R 1Selected from C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；Work as R 7In the presence of, each R 7Independently selected from halogen and C 1-C 10Alkyl.
In some embodiments, R 1Selected from phenyl, naphthalene and C 5-C 6Heteroaryl, wherein the phenyl, naphthalene and C 5-C 6Heteroaryl is optionally by 1,2 or 3 R 7Replaced group；Work as R 7In the presence of, each R 7Independently selected from-F ,-Cl ,-Br ,-I and C 1-6Alkyl.
In some embodiments, R 1Selected from following radicals:
In some embodiments, R 1Selected from following radicals:
In some embodiments, R 3Selected from C 1-C 10Alkyl, C 1-C 10Alkyl-carbonyl ,-CO- ((CH 2) n-A) m1-((CH 2) n-B) m2-R 9aWith-CO- (A- (CH 2) n) m-B-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
R 10Independently selected from C 6-C 10Aryl and-NR 8aR 8b；
R 8a、R 8bAnd R 9aIt is each independently selected from hydrogen and C 1-C 10Alkyl；
A and B are each independently selected from-O- and-NH-；
m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6；
N is 0,1 or 2.
In some embodiments, R 3Selected from C 1-C 6Alkyl, C 1-C 6Alkyl-carbonyl ,-CO- (CH 2CH 2-A) m1-(CH 2CH 2-B) m2-R 9aWith-CO- (A-CH 2CH 2) m-B-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
R 10Independently selected from phenyl, naphthalene and-NR 8aR 8b；
R 8a、R 8bAnd R 9aIt is each independently selected from hydrogen and C 1-C 6Alkyl；
A and B are each independently selected from-O- and-NH-；
m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6.
In some embodiments, R 3Selected from C 1-C 4Alkyl, C 1-C 4Alkyl-carbonyl ,-CO- (CH 2CH 2O) m1-(CH 2CH 2NH) m2-R 9aWith-CO- (OCH 2CH 2) mNH-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
R 10Independently selected from phenyl, naphthalene and-NR 8aR 8b；
R 8a、R 8bAnd R 9aIt is each independently selected from C 1-C 4Alkyl；
M and m 1It is each independently 1,2,3,4,5 or 6；
m 2It is 0,1 or 2.
In some embodiments, R 3Selected from-CO- (CH 2CH 2O) m1-(CH 2CH 2NH) m2-R 9aWith-CO- (OCH 2CH 2) mNH-R 9a；
R 9aSelected from C 1-C 4Alkyl；
M and m 1It is each independently 1,2,3,4,5 or 6；
m 2It is 0,1 or 2.
In some embodiments, R 3Selected from following radicals:
In some embodiments, R 3Selected from following radicals:
In some embodiments, the compound is selected from:
On the other hand, the disclosure provides a kind of pharmaceutical composition, the above compound containing therapeutically effective amount and one or more medicinal carrier substances and/or diluent.
In some embodiments, the carrier includes but is not limited to: aluminium oxide, aluminum stearate, lecithin, haemocyanin such as human albumin, phosphate, glycerol, sorbic acid, potassium sorbate, the partial glyceride mixtures of saturated vegetable fatty acid, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, lanolin.The diluent, also known as filler can be selected from starch, such as starch, amylum pregelatinisatum or dextrin；2) carbohydrate, such as Icing Sugar, lactose or mannitol；3) other, such as the calcium salt of microcrystalline cellulose or inorganic salts, such as sulfuric acid, bicarbonate, carbonic acid etc..
The compound of the disclosure or its pharmaceutically acceptable salt can be administered by following approach: parenteral, local, intravenous, oral, subcutaneous, intra-arterial, intradermal, in percutaneous, rectum, encephalic, peritonaeum, intranasal, intramuscular route or as inhalant.
The compound of the application or its pharmaceutically acceptable salt can be made into various suitable dosage forms according to administration route.
On the other hand, the disclosure provides aforesaid compound or pharmaceutical composition is preparing the purposes in Smoothened inhibitor.
On the other hand, the disclosure provides aforesaid compound or pharmaceutical composition in preparation for inhibiting the purposes in the active drug of Hedgehog signal path.
On the other hand, the disclosure provides above compound or pharmaceutical composition and treats the purposes in disease relevant to Hedgehog signal path excessive activation in preparation.
On the other hand, the disclosure provides a kind of active method of inhibition Smoothened comprising the step of applying a effective amount of above compound or pharmaceutical composition to subject with this need or cell.
On the other hand, the disclosure provides a kind of inhibition Hedgehog signal path active method comprising the step of applying a effective amount of above compound or pharmaceutical composition to subject with this need or cell.
On the other hand, the disclosure provides a kind of method for treating disease relevant to Hedgehog signal path excessive activation comprising the step of applying a effective amount of above compound or pharmaceutical composition to subject with this need.
On the other hand, the disclosure provides aforesaid compound or pharmaceutical composition, is used as Smoothened inhibitor.
On the other hand, the disclosure provides aforesaid compound or pharmaceutical composition, is used to inhibit Hedgehog signal path active.
On the other hand, the disclosure provides aforesaid compound or pharmaceutical composition, is used to treat disease relevant to Hedgehog signal path excessive activation.
Cell described herein is cell line or the cell from subject.
In some embodiments, the cell is cancer cell.
In some embodiments, the cell is selected from the basal cell of canceration, the medulloblast of canceration, pith mother cells cancer cell, pancreatic cancer cell, prostate gland cancer cell, liver cancer cells, colon cancer cell, small cell lung cancer cell, breast cancer cell, human rhabdomyosarcoma cells, esophageal cancer cell, stomach cancer cell, biliary tract cancer cell, multiple myeloma cells, leukaemia cell, spinal meninges oncocyte, Malignant glioma cells and melanoma cells.
Subject described herein is mammal, such as people.
In some embodiments, the disease relevant to Hedgehog signal path excessive activation is cancer.
In some embodiments, the cancer is selected from basal-cell carcinoma, medulloblast cancer, pith mother cells cancer, cancer of pancreas, prostate cancer, liver cancer, colon cancer, Small Cell Lung Cancer, breast cancer, rhabdomyosarcoma, cancer of the esophagus, gastric cancer, cancer of bile ducts, Huppert's disease, leukaemia, meningioma, glioblastoma and melanoma.
A kind of benzo [b] the thiophene-carboxamides analog derivative and its (or its) pharmaceutically acceptable salt for being designed to provide a kind of formula (I) structure of the disclosure,
Wherein:
R 1Selected from C 5-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；
R 2Selected from-H or-CH 3；
R 3Selected from hydrogen, C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 9aR 9b、-(CH 2) nOR 9a、-(CH 2) nCONR 9aR 9b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the alkyl, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, heterocyclalkylamino, aryl or heteroaryl, optional further by one or more R 10Replaced group；
R 4、R 5、R 6Selected from-H ,-F ,-Cl ,-Br ,-I and R 5、R 6It cannot simultaneously be-H；
Work as R 7In the presence of, each R 7Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 8aR 8b、-(CH 2) nOR 8a、-(CH 2) nCONR 8aR 8b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl or C 5-C 10Heteroaryl, the wherein alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl, it is optional further by one or more selected from halogen, hydroxyl, amino, cyano, nitro, replaced aldehyde radical；
R 8a, R 8b, R 9aAnd R 9bFor it is independent be hydrogen, C 1-C 10Alkyl, halogenated C 1-C 10Alkyl, C 1-C 10Alkyl amino, C 3-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl.Or R 8a/R 8bOr R 9a/R 9bThe N being connected with them is formed together 3 to 8 unit monocycles, and 3 to 8 unit monocycle is saturation or unsaturated, including with R 8a/R 8bOr R 9a/R 9bIncluding the nitrogen-atoms connected, the hetero atom of O, S or N are each independently selected from 3 to 8 unit monocycles containing one or more；
M is 0,1 or 2；
N is 0,1 or 2；
R 10Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy.
In some embodiments, R 1Optionally from following radicals but not exclusively selected from following radicals:
In some embodiments, R 3Optionally from following radicals but not exclusively selected from following radicals:
With a kind of benzo [b] thiophene-carboxamides analog derivative and its (or its) pharmaceutically acceptable salt of formula (I) structure provided by the disclosure.
Its structure is selected from:
In some embodiments, the disclosure provides a kind of benzo [b] thiophene-carboxamides analog derivative or its pharmaceutically acceptable salt with formula (I) structure,
Wherein:
R 1Selected from C 5-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；
R 2Selected from-H or-CH 3；
R 3Selected from selected from hydrogen, hydroxyl, alkoxy, C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 9aR 9b、-(CH 2) nOR 9a、-(CH 2) nCONR 9aR 9b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl, optional further by one or more R 10Replaced group；
R 4、R 5、R 6It is each independently selected from-H ,-F ,-Cl ,-Br ,-I and R 5、R 6It cannot simultaneously be-H.
In some embodiments, R 1Selected from C 5-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；
Work as R 7In the presence of, each R 7Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 8aR 8b、-(CH 2) nOR 8a、-(CH 2) nCONR 8aR 8b, carboxyl, aldehyde radical, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, C 1-C 10Alkoxy carbonyl, C 3-C 10Cyclo alkoxy carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl or C 5-C 10Heteroaryl；R 8aAnd R 8bFor it is independent be hydrogen, C 1-C 10Alkyl, halogenated C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl or R 8a、R 8bThe N being connected with them is formed together 3 to 8 unit monocycles, and 3 to 8 unit monocycle is saturation or unsaturated, including with R 8a、R 8bIncluding the nitrogen-atoms connected, the hetero atom of O, S or N are each independently selected from 3 to 8 unit monocycles containing one or more；
M is 0,1 or 2；
N is 0,1 or 2.
In some embodiments, R 1Optionally from following radicals but not exclusively selected from following radicals:
In some embodiments, R 3Selected from selected from hydrogen, hydroxyl, alkoxy, C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 9aR 9b、-(CH 2) nOR 9a、-(CH 2) nCONR 9aR 9b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl or C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl, optional further by one or more R 10Replaced group；
R 9aAnd R 9bFor it is independent be hydrogen, C 1-C 10Alkyl, halogenated C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl or R 9a、R 9bThe N being connected with them is formed together 3 to 8 unit monocycles, and 3 to 8 unit monocycle is saturation or unsaturated, including with R 9a、R 9bIncluding the nitrogen-atoms connected, the hetero atom of O, S or N are each independently selected from 3 to 8 unit monocycles containing one or more；
M is 0,1 or 2；
N is 0,1 or 2；
R 10Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy.
In some embodiments, R 3Optionally from following radicals but not exclusively selected from following radicals:
The disclosure also provides a kind of pharmaceutical composition, the pharmaceutical composition includes the free form of therapeutically effective amount or a kind of benzo [b] thiophene-carboxamides analog derivative with formula (I) structure of pharmaceutical acceptable salt and its (or its) pharmaceutically acceptable salt as active constituent and/or one or more medicinal carrier substances and/or diluent.
Pharmaceutical composition provided by the disclosure is applied in the active drug of activation Hedgehog signal path and beauty product preparation, and for adjusting cell Proliferation or differentiation, a part for treatment or cosmetic applications gives patient.
Wherein the treatment or cosmetic applications, which are selected from, adjusts the formation and reparation of nerve fiber, bone and cartilage, the growth for adjusting spermatogenesis, adjusting smooth muscle, adjusts lung, liver and other organ growths originating from primitive gut, adjusts hematopoiesis function and adjust the growth of skin and hair.
Pharmaceutical composition provided by the disclosure is also applied to inhibit the active medicine preparation of Hedgehog signal path, and for the drug for treating the disease improved by Hedgehog activity suppression, these diseases include but is not limited to cancer.
Wherein the cancer is selected from basal-cell carcinoma, medulloblast cancer, pith mother cells cancer, cancer of pancreas, prostate cancer, liver cancer, colon cancer, Small Cell Lung Cancer, breast cancer, rhabdomyosarcoma, cancer of the esophagus, gastric cancer, cancer of bile ducts, Huppert's disease, leukaemia, meningioma, glioblastoma, melanoma.
The disclosure additionally provides the preparation method of a kind of benzo [b] thiophene-carboxamides analog derivative and its (or its) pharmaceutically acceptable salt with formula (I) structure, the preparation including intermediate and disclosure compound.
Disclosure compound, its pharmaceutically acceptable salt, hydrate, solvate or combinations thereof object can be prepared by synthetic method as described herein, the compound of formula (I) structure can be used as individual isomer, racemic modification exists, and also can be used as geometric isomer presence.
Preparation method preparation well known by persons skilled in the art can be used in the compound of the disclosure.Except there is incomparable inconsistent regulation, reactions described herein under atmospheric pressure, carries out within the temperature range of about -78 DEG C to about 150 DEG C.
In order to complete the purpose of the disclosure, the disclosure adopts the following technical scheme that (following scheme is only to illustrate the disclosure, rather than be used to limit the disclosure).
Synthesis flow
The illustrative preparation method of compound 3
Using various reactions well known by persons skilled in the art, aryl side chains can be modified.The chemical method of aromatic ring and hetero-aromatic ring be it is abundant, only provide some examples of useful reaction herein.Method one:
- gram alkylated reaction is paid, some naphthenic base are replaced and the method can be used in arylmethyl substitution.
Method two:
The method can be used when replacing for some aromatic rings or heteroaromatic in Suzuki coupling reaction.
Or
Method three:
Stille coupling reaction, for some no β-hydrogen and the method can be used in the biggish substituent group of volume.
The illustrative preparation method of compound 6
Using various reactions well known by persons skilled in the art, available secondary amine and tertiary amine, R3 can be divided into three classes on the whole, alkyl and cycloalkyl group, aromatic ring and heteroaromatic and carbonyls.Likewise, the synthetic method of compound also can substantially be divided into three classes.
Method one, halogenated hydrocarbons method.
Method two, Borch reductive amination method.
Method three, N- acylation reaction
The illustrative preparation method of compound 7
Compound 7 can be obtained by Borch reductive amination process in compound 4 and compound 6
The illustrative preparation method of compound 12
Using various reactions well known by persons skilled in the art, starting material selects benzaldehyde derivative, reacts to obtain cinnamic acid derivative through Knoevenagel, then react to obtain target product with compound 11.
The illustrative preparation method of target product compound 1
By N- acylation reaction, amine (compound 7) and acyl chlorides (compound 12) generate amide, to obtain target product compound 1.
As used herein, term " C 1-C 10Alkyl " refers to the linear or branched alkyl group with 1-10 carbon atom, such as C 1-C 6Alkyl, C 1-C 4Alkyl or C 1-C 2Alkyl etc..Specific example includes but is not limited to methyl, ethyl, propyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, tert-butyl etc..
As used herein, term " C 1-C 10Alkoxy " refers to C 1-C 10The group that alkyl-O- mode is formed, wherein " C 1-C 10Described in alkyl " text as defined above.Specific example includes but is not limited to methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy, sec-butoxy, isobutoxy, tert-butoxy etc..
As used herein, term " C 1-C 10Alkylamino " refers to C 1-C 10The group that alkyl-NH- mode is formed, wherein " C 1-C 10Described in alkyl " text as defined above.Specific example includes but is not limited to methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy, sec-butoxy, isobutoxy, tert-butoxy etc..
Term " C use herein 3-C 10Naphthenic base " refers to the naphthenic base containing 3-10 ring carbons, such as C 3-C 8Naphthenic base, C 3-C 6Naphthenic base, C 5-C 10Naphthenic base, C 5-C 6Naphthenic base, C 5、C 6、C 7、C 8、C 9Or C 10Naphthenic base.Specific example includes but is not limited to: cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl etc..
Term " C use herein 3-C 10Heterocyclylalkyl " refers to the naphthenic base containing 3-10 ring members, and at least one at most 4 (such as 1,2,3 or 4) is the hetero atom selected from N, O and S, such as C in the ring members 3-C 8Heterocyclylalkyl, C 3-C 6Heterocyclylalkyl, C 5-C 10Heterocyclylalkyl, C 5-C 6Heterocyclylalkyl, C 5、C 6、C 7、C 8、C 9Or C 10Heterocyclylalkyl.Specific example includes but is not limited to: epoxy ethyl, oxocyclobutyl, pyrrolidinyl, tetrahydrofuran base, piperidyl, piperazinyl, morpholinyl, thiomorpholine base etc..
As used herein, term " C 6-C 10Aryl " refers to the monocycle containing 6-10 carbon atom, bicyclic or Ppolynuclear aromatic group, such as phenyl, naphthalene etc..
As used herein, term " C 5-C 10Heteroaryl " refers to the heteroatomic 5-10 cyclic group with armaticity that N, O and S are at least selected from containing one, such as C 5、C 6、C 7、C 8、C 9Or C 10Heteroaryl.Specific example includes but is not limited to furyl, thienyl, pyrrole radicals, thiazolyl, oxazolyl, isoxazolyl, imidazole radicals, pyrazolyl, pyridyl group, pyrimidine radicals, pyridazinyl or pyrazinyl etc..
As used herein, term " C 1-C 10Alkoxy carbonyl group " refers to C 1-C 10The group that alkoxy -C O- mode is formed, wherein " C 1-C 10Described in alkoxy " text as defined above.
As used herein, term " C 3-C 10Naphthene base carbonyl " refers to C 3-C 10The group that naphthenic base-CO- mode is formed, wherein " C 3-C 10Described in naphthenic base " text as defined above.
As used herein, term " C 5-C 10Heterocyclalkylamino " refers to C 5-C 10The group that Heterocyclylalkyl-NH- mode is formed, wherein " C 5-C 10Described in Heterocyclylalkyl " text as defined above.
As used herein, term " halogen " includes fluorine, chlorine, bromine and iodine.
" pharmaceutically acceptable salt " indicates to retain those of biological effectiveness and the property of parent compound salt.This kind of salt is included but are not limited to acid into salt, it is obtained by the free alkali of parent compound with inorganic acid or reacting for organic acid, inorganic acid includes hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid etc., organic acid includes acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, pentamethylene propionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, benzoic acid, hydroxybenzoic acid, hydroxybutyric acid, methoxy benzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene -1- sulfonic acid, naphthalene-2-sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecyl sulphate, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid or malonic acid etc..
" pharmaceutical composition " refers to one or more of compound in the disclosure or its pharmaceutically acceptable salt, solvate, hydrate or prodrug and other chemical component, such as pharmaceutically acceptable carrier, mixture.
Detailed description of the invention
Fig. 1 is the influence that the MB transplantable tumor volume subcutaneous to mouse of compound 24 is injected intraperitoneally.
Specific embodiment
Following embodiments only express the several embodiments of the disclosure, and the description thereof is more specific and detailed, and but it cannot be understood as the limitations to disclosure the scope of the patents.It should be pointed out that for those of ordinary skill in the art, under the premise of not departing from disclosure design, various modifications and improvements can be made, these belong to the protection scope of the disclosure.Therefore, the scope of protection shall be subject to the appended claims for disclosure patent.
Cell culture used herein, molecular genetics, nucleic acid chemistry, Immunology Lab operating procedure are widely used conventional steps in corresponding field.Biologic material and chemical reagent used are commercially available or are made by conventional method in that art.
Exemplary synthetic pathways
The synthesis of 1 N- of embodiment [4- (N- methylamino) cyclohexyl]-N- [[3- (4- pyridyl group) phenyl] methyl] chloro- -2 carbamide (compound 1) of 4,7- bis- fluoro- benzo [b] thiophene of -3-
1, the synthesis of trans- 4- (N-Boc-N- methylamino) cyclohexylamine
Boc acid anhydrides (1.14g, 5.25mol) and triethylamine (2.42mL, methanol (10mL) solution 17.3mmol) is added drop-wise to anti-form-1, in methanol (50mL) solution of 4- diaminocyclohexane (600mg, 5.25mmol).It reacts 40 minutes and depressurizes at 55 DEG C after being added dropwise to complete and boil off solvent, obtain oil product, product NaH 2PO 41M (pH 4.2) washing, water phase are washed with ether, are then adjusted pH to 9, are extracted with chloroform.Extract liquor is dry with anhydrous magnesium sulfate, and white product A (786mg, 70%) is obtained after reduced pressure.
By LiAlH 4(190mg, 1.0mmol) is added in THF (5mL) solution of compound A (214mg, 1.0mmol), and reaction solution is heated to reflux 2h.It is cooled to 0 DEG C after the reaction was completed, reaction solution ethyl acetate dissolves (10mL), with saturation NH 4The washing of Cl solution, separates organic layer, and water phase washs (10mL x 2) with ether.Merging organic phase is simultaneously dry with anhydrous magnesium sulfate, is concentrated to get colorless oil as product B trans- 4- (methylamino) cyclohexylamine (110mg, 86%) under organic phase reduced pressure.
The toluene solution (3mL) of benzaldehyde (117mg, 1.1mmol) is added dropwise in the toluene solution (2mL) of trans- 4- (methylamino) cyclohexylamine B (128mg, 1.0mmol), reflux 3h is heated to.Reaction solution is cooled to room temperature, and the toluene solution of Boc acid anhydrides (436mg, 2.0mmol) is added, 2h is stirred at room temperature.KHSO is added into reaction solution after the reaction was completed 4(1M), separates organic phase, and water phase washs (10mL x 2) with ether.Merge organic phase and washed with anhydrous magnesium sulfate, organic phase pressurization is concentrated to get colourless liquid C (116mg, 51%).
1H NMR(400MHz,CDCl 3):δ3.98(br s,1H,NH),3.32(br s,2H,NH+CHN),2.75-2.69(m,1H,CHN),2.72(s,3H,Me),2.05-1.97(m,2H,CH 2),1.70-1.67(m,2H,CH 2),1.54-1.20(m,4H,CH 2),1.44(s,9H,Me)；MS (ESI) m/z theoretical value C 12H 24N 2O 2(M+H) 229.1916, survey 229.1918.
2, the synthesis of chloro- -2 phosgene of 4,7- difluoro benzo [b] thiophene of 3-
Thionyl chloride (236mg, 2mmol) is added in 2,5- cinnamic acid difluoride (185mg, 1.0mmol), and reaction solution is warming up to 100 DEG C of reaction 48h.Decompression boils off solvent after the reaction was completed, with THF debris, boils off solvent and obtains chloro- -2 phosgene (compound D) of 4,7- difluoro benzo [b] thiophene (112mg, 42%) of 3-.
3, the synthesis of 3- (4- pyridyl group) benzaldehyde
At room temperature by Na 2CO 3(714mg, 6.7mmol) aqueous solution (7.0mL) be slowly added into 4- bromopyridine hydrochloride (533.4mg, in water (4.0mL) and toluene (4.8mL) solution 2.7mmol), it is added with stirring 3- carboxaldehyde radicals phenyl boric acid (431mg, 2.9mmol), Pd (PPh 3) 4(100mg, 0.086mmol).Reaction solution is warming up to 85 DEG C of reactions for 24 hours.Reaction solution is cooled to room temperature, and is extracted with dichloromethane (5mL x 4), and merging organic layer is simultaneously dry with anhydrous sodium sulfate.Decompression boils off solvent, and product separates (petrol ether/ethyl acetate=1/4) with flash column chromatography and obtains product E (420mg, 85%).
4, the synthesis of N- methyl-N- [trans- 4- [[[3- (4- pyridyl group) phenyl] methyl] amino] cyclohexyl] t-butyl carbamate
Trans- 4- (N-Boc-N- methylamino) cyclohexylamine (320mg, 1.4mmol) is added to the methanol solution of 3- (4- pyridyl group) benzaldehyde (205 mg, 1.1mmol), stirs 30 minutes at room temperature.Sodium borohydride (0.5g, 13.2mmol) is added portionwise at 0 DEG C, mixture is stirred at room temperature overnight.Saturated sodium carbonate solution is added in reaction solution, then extracts (6mLx 3) with chloroform.Merge organic layer and use anhydrous sodium sulfate, solvent under reduced pressure concentration, product silica gel column chromatography separate (methylene chloride/methanol=10/1) obtain product F (413mg, 84%).
1H NMR(400MHz,CDCl 3): δ 8.66 (dd, J=8.0,2.0Hz, 2H, ArH), 7.65 (br s, 1H, ArH), 7.56-7.40 (m, 5H, ArH), 3.90 (br s, 3H, CH 2And NH), 2.70 (s, 3H, Me), 2.50 (br s, 1H, CHN), 2.10-2.06 (m, 2H, CH 2),1.73-1.67(m,2H,CH 2),1.52-1.25(m,4H,CH 2),1.44(s,9H,Me)；MS (ESI) m/z calculated value C 24H 34N 3O 2(M+H) 396.2651, measured value 396.2658.
5, the synthesis of N- [4- (N- methylamino) cyclohexyl]-N- [[3- (4- pyridyl group) phenyl] methyl] chloro- 4,7- bis- fluoro- benzo [b] thiophene -2- carbamide of -3-
By 3- chloro- 4,7- difluoro benzo [b] thiophene -2- phosgene (395mg, 1.0mmol) it is added to triethylamine (280 μ L, 2.0mmol) and N- methyl-N- [trans- 4- [[[3- (4- pyridyl group) phenyl] methyl] amino] cyclohexyl] t-butyl carbamate (395mg, in the solution of methylene chloride (5ml) 1.0mmol), 0.5h is stirred at room temperature in reaction solution.Solvent is boiled off after the reaction was completed, obtains product G (513mg, 82%) through silica gel column chromatography separation (acetone/petroleum ether=5/1).
1H NMR(400MHz,CDCl 3):δ8.66(br s,2H,ArH),7.57(br s,1H,ArH),7.56-7.39(m,5H,ArH),7.16-7.01(m,2H,ArH),4.84(br s,1H,CH AH BAr),4.67(br s,1H,CH AH BAr),2.70-2.61(m,3H,NMe),2.01-1.67(m,4H,CH 2),1.47-1.25(m,4H,CH 2),1.44(s,9H,Me)；MS (ESI) m/z theoretical value C 33H 34ClF 2N 3O 3S, (m/z) 625.1977, measured value 625.2100.
By HCl (3N, 5mL) it is added to N- [4- (N- methyl-N-Boc amino) cyclohexyl]-N- [[3- (4- pyridyl group) phenyl] methyl] -3- chloro- 4,7- bis- fluoro- benzo [b] thiophene -2- carbamide (624mg, in methanol (5ml) solution 1.0mmol), 6h is stirred at room temperature.Reaction solution concentration obtains white solid H (458mg, 90%) through silica gel column chromatography separation (acetone/petroleum ether=5/1).
1H NMR(400MHz,CDCl 3):δ9.46(br s,2H,ArH),8.77(br s,1H,ArH),7.56-7.00(m, 6H,ArH),7.16-7.01(m,2H,ArH),4.81(br s,1H,CH AH BAr),4.69(br s,1H,CH AH BAr),4.14-4.07(m,1H,NH),3.88-3.77(m,1H,CHN),2.86-2.67(m,1H,CHN),2.51(br s,3H,Me),2.45-2.07(m,2H,CH 2),1.94-1.79(m,2H,CH 2),1.57-1.40(m,4H,CH 2)；MS (ESI) m/z theoretical value C 28H 26ClF 2N 3OS (m/z) 525.1453 surveys 525.1428.
2 3- of embodiment chloro- 4, the preparation of the fluoro- N- of 7- bis- ((1R, 4R) -4- (N- methyl -2- phenyl acetamide) cyclohexyl)-N- (3- (pyridin-4-yl) benzyl) benzo [b] thiophene-2-carboxamide derivatives (compound 3)
Phenylacetic acid (13.6mg, 0.10mmol) is dissolved in SOCl 2In (3.0mL), it is warming up to 40 DEG C of reaction 4.0h.Reaction solution is cooled to room temperature, and is dissolved in anhydrous DCM (2.5mL) after reduced pressure, is added in the anhydrous DCM (1.0mL) of compound H (52.5mg, 0.10mmol), is stirred to react 12.0h.Reaction solution concentration, through the isolated white solid I of silica gel column chromatography (43.24mg, 67.3%).
1H NMR(400MHz,CDCl3):δ8.63(br s,2H,ArH),7.57(br s,1H,ArH),7.55-7.32(m,8H,ArH),7.18-6.92(m,4H,ArH),4.84(br s,1H,CH AH BAr),4.67(br s,1H,CH AH B), Ar 4.02 (s, 2H), 2.70-2.61 (m, 3H, NMe), 2.01-1.67 (m, 4H, CH2), 1.47-1.25 (m, 4H, CH2) .MS (ESI) m/z theoretical value C 36H 32ClF 2N 3O 2S (m/z) 643.1872 surveys 643.1733.
3 N- methyl-N- ((1R of embodiment, 4R) -4- (the fluoro- N '-of the chloro- 4,7- bis- of 3- (3- (pyridin-4-yl) benzyl) benzo [b] thiophene-2-carboxamide derivatives base) cyclohexyl) carbamic acid (2- dimethylamino) ethyl ester preparation
By N, N- dimethylethanolamine (17.9mg, 0.2mmol) is dissolved in THF (mL), after addition CDI (32mg, 0.2mmol) stirs 6.0h afterwards, compound H (125.43mg is added, 0.2mmol), reaction solution is warming up to 40 DEG C of reaction 12h.Reaction solution concentration, the isolated white solid J of silica gel column chromatography (68.35mg, 53.4%)
1H NMR(400MHz,CDCl3):δ8.63(br s,2H,ArH),7.57(br s,1H,ArH),7.55-7.32(m,8H,ArH),7.18-6.92(m,4H,ArH),4.84(br s,1H,CH AH BAr),4.67(br s,1H,CH AH BAr),4.02(s,2H),3.87(d,6H),2.75-2.71(m,3H,NMe),2.01-1.67(m,8H,CH2),1.47-1.25(m,4H,CH2)；MS (ESI) m/z theoretical value C 36H 35ClF 2N 4O 3S (M+H) 641.2167 surveys 641.2178.
Following compound is made by above-mentioned route
Pharmacological activity test
1. compound and Smo targeted integration are tested
(1) Smoothened membrane filtration combines test
With transfected mouse or people Smo cDNA CHO-K1 cell be made the cell membrane containing Smo, test compound be made into graded series be put in it is spare in sky analysis plates.Cell membrane containing Smo is added in analysis plates, is added and uses buffer solution (50mM Tris-HCl, 10mM EDTA, and 5mM MgSO 4, pH 7.2) be made 1.5nM [ 3H] Hh-Ag 1.5, analysis plates are placed at 37 DEG C and are incubated for 48h.96 hole Unifilter GF/B filter plates are first incubated for 60 minutes with 0.5% polyethyleneimine and 1% fetal calf serum in advance, are washed three times with 2%HPCD distilled water solution.Culture solution in analysis plates is moved in filter plate with Unifilter-96 liquid-transfering gun, the filter plate of loading is washed three times with 2% HPCD buffer, wash away it is unbonded [ 3H] Hh-Ag1.5,60 DEG C drying 30 minutes, it is cooling.Seal bottom end, each hole adds the Microscint-O of 50 μ L, seals top, be incubated for 100 minutes-overnight.[ 3H] binding capacity of Hh-Ag1.5 obtains with TopCount (Perkin-Elmer) scintillation counter.Saturation combines number to analyze to obtain with Graphpad Prizm software.Experimental result is shown in Table 1 3-4 column.
(2) 3H-cyclopamine
Cell membrane, which is used, contains 50mM HEPES and 3mM MgCl 2, pH be 7.2 buffer solution be diluted to 0.01-0.02mg/ml.First contain to 100 μ l in the buffer solution of fluorescent ligand, the inhibitor of various concentration and protease inhibitors plus the membrane suspensions of 200 μ l, then plus 0.02% fetal calf serum to reduce non-specific binding.It is carried out on 96 orifice plates in conjunction with test.96 orifice plates are incubated for 5h at 23 DEG C reaches inhibitor in conjunction with balance, is filtered off free tagged ligand fast vacuum with the glass fiber filter (0.3% polyethyleneimine) infiltrated.The cold phosphate-buffered salt that much filtrate 0.3ml contains 0.01%Triton X-100 rinses 2 times.The fluorescence activity of much filtrate is quantitative with TopCount liquid scintillation counter.
(3) 3H-GDC-0449
2×10 6In a HEK-293 cell inoculation to the plank of 10cm, with the GeneJuice plasmid-transfected cells of 3 μ g SMO expression.After 40h, cell is transferred in 1mmol/L EDTA solution, is washed three times with PBSA after ten minutes with the PBS solution processing containing 4% paraformaldehyde.Take 96 orifice plate one, every hole refinement born of the same parents 2 × 10 5It is a, add unmarked GDC-0449 or the blank control of 50mM, then be added 5nM [ 3H]-GDC-0449,37 DEG C of incubation 1h in PBS.Cell is transferred in filter disc, is washed 6 times and dry.Radioactivity is measured with Microscint-20 scintillation solution and Topcount readout instrument.As a result it is presented with initial data presentation or after removing background with SMO-WT.
(4)BODIPY-cyclopamine
Fluorescence combine test with BODIPY FL or The cyclopamine of 558/568 label carries out, method is substantially as follows: the Chinese hamster ovary celI for expressing people or mouse SMO is added in 384 orifice plates, it is handled 15 minutes with 4% paraformaldehyde room temperature, the PBS buffer solution for containing 0.5% fetal calf serum is added after washing, then the BODIPY- cyclopamine (20nM) and test compound of fluorescent marker, 37 DEG C of incubation 4h are added.Cell is rinsed with PBS, and Hoechst 33258 is dyed, and is used The analysis of Ultra imager.
2. inhibiting the test of Hh pathway activity
(1) TM3-Gli-Luc reporter gene detection test
Test compound is dissolved with DMSO, is diluted to the solution for standby of different gradients.TM3Hh12 cell (pTA-8xGli-Luc containing Hh- response reporter gene building) is used and contains 5% horse serum, F12Ham ' the s/DMEM culture solution culture of the HEPES buffer solution (pH 7.3) of 2.5% fetal calf serum and 15mM is moved back with trypsin treatment into F12Ham ' s/DMEM (1:1) culture solution containing 5% horse serum and 15mM HEPES (pH 7.3).Cell is added in the analysis plates containing compound, is placed in 37 DEG C, 5% CO 2It is incubated for 30 minutes under environment.The Hh Ag1.5 of 1nM or 25nM is added, is incubated for 48h under identical environment.After the completion of incubation, Bright-Glo (Promega E2650) or MTS reagent (Promega G258B) is added, tests the trap under fluorescence or 492nM.IC 50Value (i.e. point of inflexion on a curve value) is shone by the luciferase that Gli drives or the absorption signal of MTS analytic approach is to the log for testing compound 10Value is done curve and is obtained, and curve is obtained with R statistical software.Experimental result is shown in Table 1 secondary series.
(2) TaqMan gene expression test
Endogenic mouse Gli and Ptch1 or people Gli and Ptch1 gene expression are expressed to obtain by rat NIH 3T3 or people's HEPM cell.The 6- orifice plate culture of EMEM culture solution culture of NIH 3T3 and the HEPM cell containing 0.5%FBS, with recombination ShhN (200ng/ml), Smo inhibitor or DMSO control is added when culture.Whole RNA is obtained with TRIzol reagent, is then extracted with chloroform, and the small-sized centrifugation column purification of RNeasy is then used.Reverse transcription is carried out with efficient cDNA Reverse Transcriptase kit.With TaqMan gene expression analysis standard measure, it is inside designated as VIC/MGB Probe.RT-PCR TaqMan Gene Expression Master Mix kit, work station are the quick RT-PCR of ABI Prism 7900HT.
(3) D473H saltant type Gli fluorescent marker is tested
C3H10T1/2 cell transfecting wild type or SMO the and Gli fluorescent marker of D473H mutation.The transfection process of DNA and GeneJuice is as follows: the GeneJuice transfection reagent of the Opti-MEM and 0.15 μ L of 3 μ L being added into empty 96 orifice plates, is incubated for 5 minutes at room temperature.The 8x-Gli1 fluorescent marker of 20ng, the pcDNA-Smo-WT of the p-RL-TK and 10ng of 1ng, pcDNA-Smo-D473H or sky pcDNA3.1 control is added, careful to mix, incubation at room temperature is added in cell after 15 minutes.The careful plank that shakes ensures to be evenly distributed, and 37 DEG C contain 5%CO 2Under the conditions of be incubated for.Culture solution changes the complete growth-promoting media of 100 μ L after transfection 6h.Test compound (0.0001-10 μM) or the DMSO control of various concentration is added in transfection afterwards for 24 hours, and culture is for 24 hours.Test compound is added and measures fluorescence activity afterwards for 24 hours.The inhibiting rate relative to blank is calculated, IC is found out 50Value.Experimental result is shown in Table 1 the 5th column.
(4)Sufu -/-Activity test
Sufu -/-L cell is inoculated into 48 orifice plates (every hole 250,000), with containing 10%FBS and 1x antibiotic/neomycin DMEM culture solution culture to fusion.Culture is for 24 hours for the Opti-MEM culture solution (being free of serum) compareed after fusion with the inhibitor containing 25 μM or DMSO.After for 24 hours, extracts RNA and then obtain cDNA.With the expression of RT-PCR analysis Gli mRNA.
(5) access Choice tests
The building of TM3-SV40 cell transfects TM3 cell with the selective pGL3-SV40-Luc plasmid of 1 μ g in the presence of neomycin and obtains, and is operated with Fugene reagent by specification.It is spare to collect remaining cell with G418 selective screening two weeks for cell.
The 293T-STF-Luc cell SuperTopflash-Luc carrier responded to Wnt transfects 293T cell in the presence of neomycin and obtains.With G418 selective screening two weeks, it is spare to collect remaining cell.In order to detect response of the inhibitor to Wnt, 384 orifice plate, one every hole is taken to contain the culture solution of 30 μ l DMEM+5%FBS, every hole adds 10000 cells.After 12h, the inhibitor solution diluted is added.Add 20 μ l Wnt culture solutions, be incubated for 48h, measures fluorescence activity with BrightGlo Fluorescence kit.
293T-NF к B-Luc cell transfects 293T cell with the Luc carrier responded to NF к B in the presence of neomycin and obtains.With G418 selective screening two weeks, it is spare to collect remaining cell.In order to detect response of the inhibitor to NF к B, 384 orifice plate, one every hole is taken to contain the culture solution of 40 μ l DMEM+5%FBS, every hole adds 10000 cells.After 12h, the inhibitor solution diluted is added, after 30 minutes plus 10 μ l are containing the culture solution for recombinating TNF α.It is incubated for and measures fluorescence activity with BrightGlo Fluorescence kit afterwards for 24 hours.
3. Compound ira vitro cell activity Proliferation Ability is tested
(1) MB proliferation test
From Ptch +/-p53 -/-The tumour that transgenic mice is got is divided into multiple segments and is passed on using nude mice.Ptch +/-p53 -/-MB cell is separated from the mouse of transplanting, with containing 2%B-27, the Neurobasal culture solution culture of 1%N2 additive and 1% penicillin/streptomycin, every hole 1x 10 in the 10cm Petri disk of Abherent 7A cell.After cell culture three days, infected with wild type or D477G mutation slow virus supernatant.Smo expression is tested with RT-PCR, proliferation experiment is operated to specifications with Click-iT EdU Cell Proliferation Assay kit.Simple operations are as follows: cell first uses the EdU of 5mM to rinse 12h, it is fixed with 4% paraformaldehyde, it is permeated with the PBS buffer solution containing 0.5%Triton X-100, it rinses, then fluorophor (vitamin C of 500nM Alexa Fluor 488azide, 1mM Cu2SO4 and 5mM) is added to be incubated with.It is rinsed after being incubated for PBS, Hoechst 33258 is dyed, ImageXpress Ultra imaging system analysis.
(2) CGNP cell proliferation test
CGNP is isolated from 4 -day-old of C57BL/6.Additive containing 2%B-27, the Sodium Pyruvate of 1mM, the Neurobasal culture solution culture of 2mM L-Glutamine and 1% penicillin/streptomycin additive, every hole cell concentration 10 are used on 384 orifice plates for secure poly-D-lysine 5, recombination ShhN (200ng/ml) and inhibitor is then added.Click-iT EdU Cell Proliferation Assay kit carries out cell proliferation experiment, and method is same as above.
(3) Gli mRNA inhibits test
HEPM cell is to obtain from human embryonic palatal mesenchymal's tissue derived from ATCC.In the case of the MEM nonessential amino acid of HEPM cell+10% fetal calf serum of minimum basic culture solution (MEM)+Earle ' s Salts+L- glutamine, 1x Sodium Pyruvate and 1x, contain 5%CO at 35 DEG C 2Under the conditions of cultivate.Cell is dispensed weekly twice with 0.05% Trypsin-EDTA solution, is then placed into the conical flask of 75cm.Cell is added in 96 orifice plates with the quantity in every hole 50000, is analyzed for Gli1RT-PCR.Next the agonist of 50nM and the inhibitor of various concentration is added.48h, which is waited, collects cell, is used for RT-PCR.
After cell is cultivated under conditions of 35 DEG C 5%, culture medium is removed, the inhibitor of 50 μ l various concentrations is added, then plus 50 μ l agonists, is incubated for 48h.The diluted agonist Hh Ag1.5 (1:1000) of 50 μ l culture mediums being added, makes the concentration 50nM of the agonist in each hole.Inhibitor is then obtained with 1000 times of culture solution dilution with the DMSO solution of 1:3 dilution 10mM.
With being collected with liquid-transfering gun and purify the cell RNA on 96 orifice plates.96 new orifice plates are taken, the reverse transcriptase of 4 μ L is previously added, above-mentioned 16 μ L of RNA solution is then added.Obtained mixed liquor is transferred on ABI96 orifice plate, is incubated at 25 DEG C 5 minutes with PCR instrument, 42 DEG C are incubated for 30 minutes, and 85 DEG C are incubated for 5 minutes.After reaction is completed, each hole adds the 40 μ L of liquid for making each hole without DNA enzymatic/RNA enzyme water of 20 μ L.Sample is stored in spare at -20 DEG C.
The quantitative expression of mRNA RT-PCR method is carried out on the quick RT-PCR instrument of Applied Biosystems 7900HT or 7900HT sequence detection system using Taqman reagent.Instrument setting procedure is as follows: cancelling ROX, 50 DEG C of 2min of first stage, 95 DEG C of 10min of second stage, 95 DEG C of 15sec of phase III, last 60 DEG C of 1min are recycled 40 times.5 kinds of solution are needed in reaction process: the primer of 2xMaster mix, Gli and betaactin react required cDNA and water.Notice that 2 × Master mix is needed comprising following component: Amplitaq Gold archaeal dna polymerase, Amperase UNG, dNTP ' s, with UTP, negative control ROX.
The data removal betaactin of real-time PCR influences.To obtain IC 50Value, needs first to deduct the background value of DMSO, calculates relative to not having inhibiting rate of the inhibiting containing only agonist.IC 50Value is calculated with XLfit4using Fit Designer Dose Response Model 202.
(4) A549 cell proliferation experiment
By the A549 cell suspending liquid of logarithmic growth phase, it is diluted to 4 × 10 4-5×10 4A/mL is inoculated in 96 orifice plates (100 hole μ L/).In 37 DEG C, 5%CO 2Under the conditions of be incubated for 8h after, the chemical compound diluted liquid (culture medium dilution) of 100 μ L various concentrations is added in every hole, and each concentration sets three multiple holes.After being incubated for 48h, the MTT dyeing liquor of 10 μ L0.5% is added in every hole, continues to be incubated for 4h, and rear centrifugation discards supernatant liquid, 150 μ L DMSO are added in every hole, is incubated for 5-10min in 37 DEG C of shaking tables.The OD value at 570nM is measured with microplate reader, and calculates the inhibiting rate and IC of compound 50Value.Experimental result is shown in Table 1 the 7th column.
4. inhibiting the foundation of the animal model of tumor promotion in compound animals body
(1) compound 24 inhibits to test to subcutaneous MB transplantable tumor
Take 1.0 × 10 6Or 5.0 × 10 6A Ptch obtained from tumor sample +/-Hic -/-Mouse MB cell inoculates arrive nude mice or nude rat right abdomen respectively.7 days beginning medicine feeds after transplanting are grouped before medicine feed at random according to tumor size, are 250mm with each group of average tumor size 3It is advisable.Daily medicine feed twice, totally 13 days.The gross tumor volume and weight of every group of mouse need to record 2-3 times weekly, for post analysis.Medicine feed dosage is changed at any time with weight, and the control between different treatment groups is calculated with ANOVA (Tukey) rank sum test method.Experimental result is as shown in Figure 1.
(2) cancer of pancreas transplantation model is tested
E3LZ10.7 cell subcutaneous injection enters in male CD1 nude mouse, and tumour is won after 2-4 weeks, is cut into about 1mm 3Fritter.6-8 weeks male CD1 nude mice is anaesthetized with anaesthetic gases, is opened under the rib of 1cm on the left of abdomen, until that can see spleen and pancreas adjunct, an osculum is opened on pancreas and is implanted into ready tumour and sutures.
In L3.6pl group, 10 6A cell is suspended in PBS/Matrigel (1:1) mixed liquor of 50 μ L, is directly injected into mice pancreatic.After 21 days, with the volume of ultrasonic measurement tumour.Using tumor size as standard, be equally divided into four groups: control, cyclopamine (25mg/kg feed addition is fed, once two days), gemcitabine (100mg/kg, injection in every four days are primary) and drug combination group are administered continuously 30 days.L3.6pl group is only needed to cyclopamine 15 days, 50mg/kg/d.
After 30 days, mouse is put to death, checks kidney, liver, spleen, small intestine, the tumour spread condition of peritonaeum and lung, and saved with dipped into formalin.Tumor sample, which is rapidly frozen, is used for RNA analysis.In addition to directly observing, every mouse should randomly select kidney, liver, spleen, small intestine, peritonaeum and lung tissue for microscopy, and whether there is or not micrometastasis (micrometastases) for observation.Regional nodes' sample is obtained, is made into and is sliced and checks whether generation lesion.L3.6pl group only needs to observe the lesion situation of liver and peritonaeum.
5. drug metabolism is tested in compound animals body
(1) single dose bioavailability and pharmacokinetic trial in Mice Body
Compound is dissolved in the solution being made by the 10%ETPGS+50%D5W of 5%NMP+10%PEG400+35%, male C 57 BL mouse (2 each time points) tail vein injection (1mg/kg, 0.2mg/mL) or stomach-filling (10mg/kg, 1mg/ml).Set time heart puncturing extracting blood sample (2 each time points) after administration for 24 hours.Centrifugal separation plasma, with the concentration of compound in HPLC/MS/MS measurement blood plasma.Relevant plasma pharmacokinetics constant such as clearance rate, volume of distribution and half-life period are calculated by non-compartment model.
(2) single dose bioavailability and pharmacokinetic trial in rat body
Sprague-Dawley tail vein injection (1mg/kg, by 1.5eq HCl, 20%captisol is dissolved in the compound solution for the 1mg/mL that 8 buffer solution of pH is made into, 2 every time) or stomach-filling (10mg/kg, 0.5% sodium carboxymethylcellulose suspension, every time 3) method administration.According to regular time blood sampling after for 24 hours, it is centrifuged to obtain blood plasma.The concentration of compound is measured with HPLC/MS/MS in blood plasma, relevant plasma pharmacokinetics constant such as clearance rate, and volume of distribution and half-life period are calculated by non-compartment model.
(3) single dose bioavailability and pharmacokinetic trial in dog body
Compound is dissolved in 20%captisol, is made into the solution of 0.4mg/ml, male beagle dogs (3) single dose injection (0.1mg/kg) of fasting or oral (0.3mg/kg).According to regular time blood sampling after for 24 hours, it is centrifuged to obtain blood plasma.The concentration of compound is measured with HPLC/MS/MS in blood plasma, relevant plasma pharmacokinetics constant such as clearance rate, and volume of distribution and half-life period are calculated by non-compartment model.
6. compound solubility is tested
Compound is dissolved with DMSO, the mother liquor that concentration is 100mg/mL is made into, is stored under the conditions of 4 DEG C.The standard solution for being successively 10,1,0.1,0.01mg/mL with chromatography methanol dilution；LC-MS measures the peak area under various concentration, establishes standard curve.10 μ L mother liquors are taken to be added in 1mL pure water, the saturation solubility of compound is calculated in ultrasonic 0.5h, the absorption peak area of measurement compound in water.Experimental result is shown in Table 1 the 6th column.
7. Compound half-life measures
It takes pre-prepared compound testing solution (to prepare) 480 μ L according to 79:1 ratio by the particle liquid solution and 0.1mM compound solution of 0.125mg/mL to be added in 3 centrifuge tubes (parallel test 3 times), the oscillation incubation at 37 DEG C is separately added into after 120 μ L 5mM NADPH, according to time point 5min, 15min, 30min, the every 100 μ L of sub-sampling of 60min, the cold acetonitrile extraction that 600 μ L are added are gone out reaction, are then centrifuged 5min in 4 DEG C, the centrifuge of 12000rpm.Reaction of going out is extracted for the cold acetonitrile that 0min sample is 600 μ L of sampling 100 μ L addition, then adds 60 μ L NADPH solution again, then be centrifuged under identical condition.Supernatant liquor 100 μ L LC/MS detection compound concentration is taken, curve is then done according to time and compound concentration percentage, half-life period calculates according to first order rate constant.T 1/2=0.693/k, k are log percent concentration and time to do the slope that curve obtains.Experimental result is shown in Table 1 the 6th column.
Pharmacological activity test result
According to above-mentioned activity determination method, disclosure compound is determined to the combination activity of SMO and to the activity of 9 kinds of common Lines, the active testing result of part of compounds is as shown in the table:
1 disclosure compound pharmacological activity test result of table
* TM3-GLi-Luc data: greater than 500nM is D；Being greater than 300 less than 500 is C；Being greater than 100nM less than 300 is B；It is A less than 100nM.

Claims (15)

  1. Formula (I) compound or its pharmaceutically acceptable salt,
    Wherein:
    R 1Selected from C 5-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；
    R 2Selected from-H and-CH 3；
    R 3Selected from-H, hydroxyl, alkoxy, C 1-C 10Alkoxy carbonyl group, C 1-C 10Alkyl, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 9aR 9b、-(CH 2) nOR 9a、-(CH 2) nCONR 9aR 9b、-CO-((CH 2) n-A) m1-((CH 2) n-B) m2-R 9a、-CO-(A-(CH 2) n) m-B-R 9a, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl are optionally further by one or more R 10Replaced group；
    R 4、R 5、R 6It is each independently selected from-H ,-F ,-Cl ,-Br and-I, and R 5And R 6It cannot simultaneously be-H；
    Work as R 7In the presence of, each R 7Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, C 1-C 10Alkyl, alkoxy, C 3-C 10Naphthenic base, C 3-C 10Heterocyclylalkyl ,-(CH 2) nNR 8aR 8b、-(CH 2) nOR 8a、-(CH 2) nCONR 8aR 8b, carbonyl, C 1-C 10Alkyl-carbonyl, C 3-C 10Naphthene base carbonyl, amino, C 1-C 10Alkylamino, C 5-C 10Heterocyclalkylamino, C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the alkyl, alkoxy, naphthenic base, Heterocyclylalkyl, carbonyl, alkyl-carbonyl, naphthene base carbonyl, amino, ring type amidogen, heterocyclalkylamino, aryl or heteroaryl are optionally further selected from replaced the groups of halogen, hydroxyl, amino, cyano, nitro and aldehyde radical by one or more；
    R 8a, R 8b, R 9aAnd R 9bIt is each independently selected from hydrogen, C 1-C 10Alkyl, halogenated C 1-C 10Alkyl, C 1-C 10Alkyl amino, C 3-C 10Naphthenic base, C 5-C 10Heterocyclylalkyl and C 6-C 10Aryl；Or R 8a/R 8bOr R 9a/R 9bThe N being connected with them is formed together 3 to 8 unit monocycles, and 3 to 8 unit monocycle is saturation or unsaturated, including with R 8a/R 8bOr R 9a/R 9bIncluding the nitrogen-atoms connected, the hetero atom of O, S or N are each independently selected from 3 to 8 unit monocycles containing one or more；
    R 10Independently selected from halogen, hydroxyl, sulfydryl, cyano, nitro alkyl, alkoxy, C 6-C 10Aryl and-NR 8aR 8b；
    A and B are each independently selected from-O- and-NH-；
    m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6；
    N is 0,1 or 2.
  2. Compound described in claim 1 or its pharmaceutically acceptable salt, in which:
    R 1Selected from C 6-C 10Aryl and C 5-C 10Heteroaryl, wherein the naphthenic base, Heterocyclylalkyl, aryl and heteroaryl are optionally by 1,2 or 3 R 7Replaced group；Work as R 7In the presence of, each R 7Independently selected from halogen and C 1-C 10Alkyl；
    Preferably, R 1Selected from phenyl, naphthalene and C 5-C 6Heteroaryl, wherein the phenyl, naphthalene and C 5-C 6Heteroaryl is optionally by 1,2 or 3 R 7Replaced group；Work as R 7In the presence of, each R 7Independently selected from-F ,-Cl ,-Br ,-I and C 1-6Alkyl；
    Preferably, R 1Selected from following radicals:
    Preferably, R 1Selected from following radicals:
  3. Compound of any of claims 1 or 2 or its pharmaceutically acceptable salt, in which:
    R 3Selected from C 1-C 10Alkyl, C 1-C 10Alkyl-carbonyl ,-CO- ((CH 2) n-A) m1-((CH 2) n-B) m2-R 9aWith-CO- (A- (CH 2) n) m-B-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
    R 10Independently selected from C 6-C 10Aryl and-NR 8aR 8b；
    R 8a、R 8bAnd R 9aIt is each independently selected from hydrogen and C 1-C 10Alkyl；
    A and B are each independently selected from-O- and-NH-；
    m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6；
    N is 0,1 or 2；
    Preferably, R 3Selected from C 1-C 6Alkyl, C 1-C 6Alkyl-carbonyl ,-CO- (CH 2CH 2-A) m1-(CH 2CH 2-B) m2-R 9aWith-CO- (A-CH 2CH 2) m-B-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
    R 10Independently selected from phenyl, naphthalene and-NR 8aR 8b；
    R 8a、R 8bAnd R 9aIt is each independently selected from hydrogen and C 1-C 6Alkyl；
    A and B are each independently selected from-O- and-NH-；
    m、m 1And m 2It is each independently 0,1,2,3,4,5 or 6；
    Preferably, R 3Selected from C 1-C 4Alkyl, C 1-C 4Alkyl-carbonyl ,-CO- (CH 2CH 2O) m1-(CH 2CH 2NH) m2-R 9aWith-CO- (OCH 2CH 2) mNH-R 9a, wherein the alkyl and alkyl-carbonyl are optionally further by one or more R 10Replaced group；
    R 10Independently selected from phenyl, naphthalene and-NR 8aR 8b；
    R 8a、R 8bAnd R 9aIt is each independently selected from C 1-C 4Alkyl；
    M and m 1It is each independently 1,2,3,4,5 or 6；
    m 2It is 0,1 or 2；
    Preferably, R 3Selected from following radicals:
  4. Compound described in claim 1 or its pharmaceutically acceptable salt, wherein the compound is selected from:
  5. Compound described in claim 1 or its pharmaceutically acceptable salt, wherein the compound is selected from:
  6. Pharmaceutical composition contains compound described in claim 1 to 5 any one of therapeutically effective amount as active constituent and one or more medicinal carrier substances and/or diluent.
  7. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6 are preparing the purposes in Smoothened inhibitor.
  8. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6 are in preparation for inhibiting the purposes in the active drug of Hedgehog signal path.
  9. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6 treat the purposes in disease relevant to Hedgehog signal path excessive activation in preparation, preferably, the disease is cancer, preferably, the cancer is selected from basal-cell carcinoma, medulloblast cancer, pith mother cells cancer, cancer of pancreas, prostate cancer, liver cancer, colon cancer, Small Cell Lung Cancer, breast cancer, rhabdomyosarcoma, cancer of the esophagus, gastric cancer, cancer of bile ducts, Huppert's disease, leukaemia, meningioma, glioblastoma and melanoma.
  10. A kind of active method of inhibition Smoothened comprising the step of applying compound described in a effective amount of claim 1 to 5 any one or pharmaceutical composition as claimed in claim 6 to subject with this need or cell.
  11. A kind of active method of inhibition Hedgehog signal path comprising the step of applying compound described in a effective amount of claim 1 to 5 any one or pharmaceutical composition as claimed in claim 6 to subject with this need or cell.
  12. A method for the treatment of disease relevant to Hedgehog signal path excessive activation, it includes the steps that applying compound described in a effective amount of claim 1 to 5 any one or pharmaceutical composition as claimed in claim 6 to subject with this need, preferably, the disease is cancer, preferably, the cancer is selected from basal-cell carcinoma, medulloblast cancer, pith mother cells cancer, cancer of pancreas, prostate cancer, liver cancer, colon cancer, Small Cell Lung Cancer, breast cancer, rhabdomyosarcoma, cancer of the esophagus, gastric cancer, cancer of bile ducts, Huppert's disease, leukaemia, meningioma, glioblastoma and melanoma.
  13. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6 are used as Smoothened inhibitor.
  14. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6 are used to inhibit Hedgehog signal path active.
  15. Compound described in claim 1 to 5 any one, pharmaceutical composition as claimed in claim 6, it is used to treat disease relevant to Hedgehog signal path excessive activation, preferably, the disease is cancer, preferably, the cancer is selected from basal-cell carcinoma, medulloblast cancer, pith mother cells cancer, cancer of pancreas, prostate cancer, liver cancer, colon cancer, Small Cell Lung Cancer, breast cancer, rhabdomyosarcoma, cancer of the esophagus, gastric cancer, cancer of bile ducts, Huppert's disease, leukaemia, meningioma, glioblastoma and melanoma.
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