CN104230897B - Benzimidazolyl-2 radicals-piperazine heterocycle compound, its pharmaceutical composition and its production and use - Google Patents

Benzimidazolyl-2 radicals-piperazine heterocycle compound, its pharmaceutical composition and its production and use Download PDF

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CN104230897B
CN104230897B CN201310728778.XA CN201310728778A CN104230897B CN 104230897 B CN104230897 B CN 104230897B CN 201310728778 A CN201310728778 A CN 201310728778A CN 104230897 B CN104230897 B CN 104230897B
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fluoro
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benzimidazole
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CN104230897A (en
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樊兴
秦继红
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Shanghai Huilun Pharmaceutical Co ltd
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SHANGHAI HUILUN TECHNOLOGY Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

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Abstract

Benzimidazolyl-2 radicals of the present invention-piperazine heterocycle compound, its pharmaceutical composition and its production and use, relate to trifluoromethyl pyrimidine compound, its preparation method and in application pharmaceutically.Concrete, the present invention relates to the new cyanopyrimidine compound shown in a kind of formula (33), its preparation method and the pharmaceutical composition containing this compound and it is as the therapeutic agent purposes especially as poly-(ADP-ribose) polymerase (PARP) inhibitor.

Description

Benzimidazolyl-2 radicals-piperazine heterocycle compound, its pharmaceutical composition and its production and use
The application is the application number submitted on June 17th, 2013 is 201310240069.7, and denomination of invention is the divisional application of " benzimidazolyl-2 radicals-piperazine compounds, its pharmaceutical composition and its production and use ".
Technical field
The present invention relates to benzimidazolyl-2 radicals-piperazine heterocycle compound, its preparation method and the pharmaceutical composition containing this compound and it is as therapeutic agent with as the purposes gathering (ADP-ribose) polymerase (PARP) inhibitor.
Background technology
Chemotherapeutics and ionizing radiation treatment are two kinds of common methods for the treatment of cancer.Both Therapeutic Method all can bring out DNA single chain and/or double-strand break and then produce cytotoxic effect, target tumor due to chromosome damage thus death.An important results as response DNA damage signal is that cell cycle regulating site signal is activated, and its object is to protection cell and does not carry out mitosis thus avoiding cell injury when DNA damage.In most of the cases, tumor cell has significantly high appreciation rate while showing cell cycle regulating site signal defect.It can therefore be concluded that tumor cell exists specific DNA repair mechanism, it is possible to quickly response reparation regulate relevant chromosome damage to propagation, so that himself surviving cytotoxic effect the maintenance survival of some medicines.
In clinical practice, valid density or the treatment radiant intensity of chemotherapeutics can resist these DNA repair mechanisms, it is ensured that the fragmentation effect to target tumor.But, treatment can be produced tolerance effect by strengthening its DNA damage repair mechanism by tumor cell, so as to survive from fatal DNA damage.In order to overcome the toleration of generation, it usually needs increasing the dosage of medicine or improve radiant intensity, the normal structure near focus will be had a negative impact by this way, so that therapeutic process is with serious untoward reaction, and then increases Operative risk.Meanwhile, ever-increasing toleration will reduce therapeutic effect, it can therefore be concluded that by the adjustment to DNA damage signal repair mechanism, it is possible in the way of tumor cell specific, realize the Cytotoxic raising to DNA damage medicament.
The PARPs(Poly(ADP-ribose being feature with poly-adenosine diphosphate-ribosylating activity) polymerases), constitute the superfamily of 18 kinds of cell ribozyme nucleus matter enzymes.This poly-adenosine diphosphate-ribosylation can regulate catalysis activity and the protein-protein interaction of destination protein, and many basic bioprocesss are regulated and controled, and repairs including DNA, and cell death, Genome stability is also associated.
PARP-1 activity accounts for the 80% of total cell PARP activity, and it and the PARP-2 the most close with it become the member possessing DNA plerosis lesion capability in PARP family jointly.As induction apparatus and the signal protein of DNA damage, PARP-1 can quickly detect and be bonded directly to DNA damage site, and induced aggregation DNA repairs required multiple protein afterwards, and then makes DNA damage be repaired.When the PARP-1 in cell lacks, PARP-2 can substitute PARP-1 and realize the reparation of DNA damage.Research shows, compared with normal cell, the expression in solid tumor of the PARPs albumen generally strengthens.In addition, DNA Related to repair gene is lacked to the tumor (such as breast tumor and ovarian cancer) of (such as BRCA-1 or BRCA-2), show the extreme sensitivity to PARP-1 inhibitor, this show PARP inhibitor as single dose in this potential use being known as in three negative breast cancer for the treatment of.Simultaneously as DNA damage repair mechanism is tumor cell reply chemotherapeutics and the ionizing radiation treatment resistance to main mechanism the affected of generation, therefore PARP-1 is considered as the Effective target site exploring new cancer treatment method.
The PARP inhibitor of early development design is using the nicotiamide of the NAD as PARP catalytic substrate as template, develops its analog.These inhibitor as the competitive inhibitor of NAD, and the catalytic site that NAD competes PARP, and then stop the synthesis of poly-(ADP-ribose) chain.PARP under not having poly-(ADP-ribosylation) to modify cannot disintegrate down from DNA damage site, will cause that other protein participating in repairing cannot be introduced into injury site, and then can not perform repair process.Therefore, under the effect of cytotoxic drug or radiation, the existence of PARP inhibitor makes tumor cell impaired for DNA finally dead.
Additionally, the NAD being consumed as PARP catalytic substrate, be in cell synthesis ATP process requisite therefore.Under high PARP activity level, intracellular NAD level can be remarkably decreased, and then affects the ATP level in born of the same parents.Owing to the ATP content in born of the same parents is not enough, cell cannot realize the ATP programmed cell death process relied on, and can only turn to this special apoptotic process downright bad.In downright bad process, substantial amounts of inflammatory factor can be released, thus other organs and tissue are produced toxic action.Therefore, PARP inhibitor can be used for the multiple disease that treatment is relevant with this mechanism, including neurodegenerative diseases (such as senile dementia, Huntington chorea, parkinson disease), diabetes, ischemia or the complication in Ischemia-Reperfusion Injury, such as myocardial infarction and acute renal failure, blood circulation diseases, such as septic shock, and diseases associated with inflammation, such as chronic rheumatism etc..
Summary of the invention
An object of the present invention is in that to provide a kind of new benzimidazolyl-2 radicals-piperazine heterocycle compound and derivant thereof, and their tautomer, enantiomer, diastereomer, raceme and pharmaceutically useful salt, and metabolite and metabolite precursor or prodrug.
The two of the purpose of the present invention are in that to provide the pharmaceutical composition using described benzimidazolyl-2 radicals-piperazine heterocycle compound as active component.
The three of the purpose of the present invention are in that the preparation method providing above-mentioned benzimidazolyl-2 radicals-piperazine heterocycle compound.
The four of the purpose of the present invention are in that to provide above-mentioned benzimidazolyl-2 radicals-piperazine heterocycle compound application in medicine.
As the benzimidazolyl-2 radicals-piperazine heterocycle compound of first aspect present invention, it is the compound shown in formula (I):
Wherein, in logical formula I, R is hydrogen or halogen；
X, Y, Z one of them be nitrogen, all the other are hydrocarbon for hydrocarbon or X, Y, Z；
R1For hydrogen, C1-C6Alkyl, methoxyl group, trifluoromethyl, halogen, nitro, cyano group, CONR2R3And NR2R3；
R2For hydrogen, C1-C6Alkyl；
R3For hydrogen, C1-C6Alkyl, C3-C6Cycloalkyl or NR2R3Cyclization forms morpholine base, nafoxidine base and piperidyl together.
It is further preferred that the structure provided by the invention such as compound shown in logical formula I, wherein: R is hydrogen or fluorine；
X, Y, Z one of them be nitrogen, all the other are hydrocarbon for hydrocarbon or X, Y, Z；
R1For hydrogen, C1-C4Alkyl, methoxyl group, trifluoromethyl, fluorine, nitro, cyano group, CONR2R3And NR2R3；
R2For hydrogen, C1-C4Alkyl；
R3For hydrogen, C1-C4Alkyl, C3-C6Cycloalkyl or NR2R3Cyclization forms morpholine base, nafoxidine base together.
Most preferably, the logical compound shown in formula I of the present invention is following compound (1)～(37):
Described logical formula I compound is the mixture of any one or arbitrarily both or the three in enantiomer, diastereomer, conformer.
Described logical formula I compound is pharmaceutically acceptable derivant.
Logical formula I compound of the present invention can exist as a pharmaceutically acceptable salt form.
Pharmaceutically acceptable salt of the present invention is the logical hydrochlorate of formula I compound, sulfate, phosphate, acetate, trifluoroacetate, mesylate, fluoroform sulphonate, tosilate, tartrate, maleate, fumarate, succinate or malate.
In a preferred embodiment of the invention, the benzimidazolyl-2 radicals of described logical formula I-piperazine heterocycle compound is 2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide compounds and its officinal salt.
As the preparation method of the compound shown in the logical formula I of second aspect present invention, its reaction equation is as follows:
Wherein, R and R1As defined above；Specifically comprise the following steps that
Step 1): 2,3-diamidogen yl benzoic acid methyl ester of replacement and carbonyl dimidazoles ring-closure reaction, obtains 2-oxo-2 replaced, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (II)；
Step 2): 2-oxo-2 of the replacement that step 1) obtains, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (II) and phosphorus oxychloride carry out chlorination reaction, obtain chloro-1 hydrogen of the 2--benzimidazole-4-methyl formate (III) replaced；
Step 3): in the presence of a base, by step 2) chloro-1 hydrogen of the 2--benzimidazole-4-methyl formate (III) of replacement and the piperazine that obtain carry out nucleophilic substitution, obtains 2-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (IV) replaced；
Step 4): the aminolysis reaction of ester group occurs 2-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (IV) of replacement step 3) obtained in methanolic ammonia solution, obtains 2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (V) replaced；
Step 5): 2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (V) of the replacement that step 4) is obtained and acid occur coupling reaction or with aldehyde generation reductive amination process, generate logical compound shown in formula I.
As the Pharmaceutical composition of third aspect present invention, comprise logical formula I compound and one or more medicinal carrier substances and/or the diluent of the therapeutically effective amount constituting active component.Or comprise logical formula I compound and pharmaceutically acceptable carrier, excipient or the diluent of the therapeutically effective amount constituting active component.
As the Pharmaceutical composition of third aspect present invention, comprise pharmaceutically acceptable derivant and one or more medicinal carrier substances and/or the diluent of the logical formula I compound of the therapeutically effective amount constituting active component.Or comprise pharmaceutically acceptable derivant and pharmaceutically acceptable carrier, excipient or the diluent of the logical formula I compound of the therapeutically effective amount constituting active component.
As the Pharmaceutical composition of third aspect present invention, comprise pharmaceutically acceptable salt and one or more medicinal carrier substances and/or the diluent of the logical formula I compound of the therapeutically effective amount constituting active component.Or comprise pharmaceutically acceptable salt and pharmaceutically acceptable carrier, excipient or the diluent of the logical formula I compound of the therapeutically effective amount constituting active component.
Described pharmaceutical composition makes tablet, capsule, aqueous suspension, Oil suspensions, dispersible powder, granule, lozenge, Emulsion, syrup, ointment, ointment, suppository or injection.
In described pharmaceutical composition, described logical formula I compound exists in a free form.
As the application of fourth aspect present invention, it it is wherein the application in the disease medicament that preparation treatment improves because PARP activity suppresses of the described logical formula I compound.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically acceptable derivant of described logical formula I compound treats the application in the disease medicament improved because PARP activity suppresses in preparation.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically useful salt of described logical formula I compound treats the application in the disease medicament improved because PARP activity suppresses in preparation.
As the application of fourth aspect present invention, it it is wherein the application in the disease medicament that preparation treatment improves because PARP activity suppresses of the described pharmaceutical composition.
The described disease improved because PARP activity suppresses is angiopathy, septic shock, ischemic injuries, neurotoxicity, hemorrhagic shock, inflammatory diseases or multiple sclerosis.
As the application of fourth aspect present invention, it is wherein that described logical formula I compound is being prepared for the application in the ancillary drug of oncotherapy.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically acceptable derivant of described logical formula I compound is being prepared for the application in the ancillary drug of oncotherapy.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically useful salt of described logical formula I compound is being prepared for the application in the ancillary drug of oncotherapy.
As the application of fourth aspect present invention, it is wherein that described pharmaceutical composition is being prepared for the application in the ancillary drug of oncotherapy.
As the application of fourth aspect present invention, it is wherein that described logical formula I compound strengthens the application in the medicine of radiotherapy in preparation for tumor.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically acceptable derivant of described logical formula I compound strengthens the application in the medicine of radiotherapy in preparation for tumor.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically useful salt of described logical formula I compound strengthens the application in the medicine of radiotherapy in preparation for tumor.
As the application of fourth aspect present invention, it is wherein that described pharmaceutical composition strengthens the application in the medicine of radiotherapy in preparation for tumor.
As the application of fourth aspect present invention, it is wherein that described logical formula I compound is being prepared for the application in the medicine of chemotherapy of tumors.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically acceptable derivant of described logical formula I compound is being prepared for the application in the medicine of chemotherapy of tumors.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically useful salt of described logical formula I compound is being prepared for the application in the medicine of chemotherapy of tumors.
As the application of fourth aspect present invention, it is wherein that described pharmaceutical composition is being prepared for the application in the medicine of chemotherapy of tumors.
As the application of fourth aspect present invention, it is wherein that described logical formula I compound lacks the application in the medicine of the individuation treatment of cancer that homologous recombination (HR) dependent DNA double chain interruption (DSB) is repaired in preparation.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically acceptable derivant of described logical formula I compound lacks the application in the medicine of the individuation treatment of cancer that homologous recombination (HR) dependent DNA double chain interruption (DSB) is repaired in preparation.
As the application of fourth aspect present invention, it is wherein that the pharmaceutically useful salt of described logical formula I compound lacks the application in the medicine of the individuation treatment of cancer that homologous recombination (HR) dependent DNA double chain interruption (DSB) is repaired in preparation.
As the application of fourth aspect present invention, it is wherein that described pharmaceutical composition lacks the application in the medicine of the individuation treatment of cancer that homologous recombination (HR) dependent DNA double chain interruption (DSB) is repaired in preparation.
As preferably, described cancer is the cancer of the cancerous cell being lowered relative to normal cell by the ability of the DSB of HR DNA plerosis containing one or more or losing.
As preferably, described cancer is the cancer with BRCA-1 or BRCA-2 defect, mutant phenotype.
As preferably, described cancer is breast carcinoma, ovarian cancer, cancer of pancreas or carcinoma of prostate.
In order to check compound provided by the invention for the exposure level of PARP enzyme, the test of biochemistry level enzymatic activity is adopted to determine the various compounds of the present invention activity to PARP enzyme.
PARP is a kind of posttranscriptional modification enzyme, DNA damage can activate this enzyme, PARP catalytic process in vivo is mainly a kind of NAD poly(ADP-ribose relied on) process, its substrate mainly includes some nucleoprotein of PARP, histone is one of which, the present invention is by measuring PARP under NAD effect to being coated in Histonepoly(ADP-ribose in 96 orifice plates) degree, measure PARP activity, after correspondingly measuring PARP inhibitor effect, PARP is active, thus evaluating this compounds suppression degree to PARP activity.
Detailed description of the invention
Unless stated to the contrary, following use term in the specification and in the claims has following implication.
In the present invention, term " C1-C6Alkyl " refer to that there is straight or branched part the saturated monovalent hydrocarbon containing 1 to 6 carbon atom.The example of this type of group includes but not limited to methyl, ethyl, propyl group, isopropyl, normal-butyl, isobutyl group and the tert-butyl group.
Term " halogen " and " halo " refer to F, Cl, Br, I.
" pharmaceutically acceptable salt " represents those salt of biological effectiveness and the character retaining parent compound.This kind of salt includes:
(1) salt is become with acid, react by the free alkali of parent compound with mineral acid or organic acid and obtain, mineral acid includes hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, Metaphosphoric acid, sulphuric acid, sulfurous acid and perchloric acid etc., organic acid includes acetic acid, propanoic acid, acrylic acid, oxalic acid, or (L) malic acid (D), fumaric acid, maleic acid, hydroxy benzoic acid, gamma-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethyl sulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, mandelic acid, succinic acid or malonic acid etc..
(2) salt that the acid proton in parent compound is replaced by metal ion or generates with organic base ligand compound it is present in, metal ion is alkali metal ion, alkaline-earth metal ions or aluminium ion such as, and organic bases is ethanolamine, diethanolamine, triethanolamine, trometamol, N-METHYL-ALPHA-L-GLUCOSAMINE etc. such as.
" pharmaceutical composition " refers to one or more or its pharmaceutically acceptable salt, solvate, hydrate or the prodrug in the compound in the present invention and other chemical composition, for instance pharmaceutically acceptable carrier, mixing.The purpose of pharmaceutical composition is to promote that administration is to the process of animal.
" pharmaceutical carrier " refers to and organism does not cause obvious zest and does not disturb the non-active ingredient in the biological activity of given compound and the pharmaceutical composition of character, for instance but be not limited to: calcium carbonate, calcium phosphate, various sugar (such as lactose, mannitol etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylate copolymer or methacrylate polymer, gel, water, Polyethylene Glycol, propylene glycol, ethylene glycol, Oleum Ricini or castor oil hydrogenated or many ethoxy aluminium Oleum Ricini, Oleum sesami, Semen Maydis oil, Semen arachidis hypogaeae wet goods.
In aforesaid pharmaceutical composition, except including pharmaceutically acceptable carrier, it is additionally may included in adjuvant conventional in medicine (agent), for instance: antibacterial agent, antifungal, antimicrobial, preservative, toner, solubilizing agent, thickening agent, surfactant, chelating agent, protein, aminoacid, fat, saccharide, vitamin, mineral, trace element, sweeting agent, pigment, essence or their combination etc..
The invention discloses the application as poly-(ADP-ribose) AG14361 of a kind of compound and this compound, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention.Method and the application of the present invention are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention.
Below in conjunction with embodiment, the present invention is expanded on further:
Preparation embodiment
Embodiment 1
The preparation of compound (1): 2-(4-(pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide, concrete reaction equation is as follows:
The preparation of step 1:2-oxo-2,3-dihydro-1 hydrogen-benzimidazole-4-methyl formate
At one dissolved with 2,3-diamidogen yl benzoic acid methyl ester (0.8g, anhydrous tetrahydrofuran solution (20mL) 4.8mmol) adds carbonyl dimidazoles (1.56g, 9.6mmol), it is warming up to backflow, cools down after reacting 8 hours, removal of solvent under reduced pressure, residue separates (petroleum ether: ethyl acetate=5:1) through rapid column chromatography and obtains faint yellow solid compound a: 2-oxo-2,3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (0.3g, yield 33%).MS(ESI)m/z:[M+H]+=193。
The preparation of chloro-1 hydrogen of step 2:2--benzimidazole-4-methyl formate
By compound a: 2-oxo-2,3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (1.1g, 5.7mmol) add phosphorus oxychloride (8mL), it is warming up to backflow, cool down after reacting 8 hours, removal of solvent under reduced pressure, residue separates (petroleum ether: ethyl acetate=5:1) through rapid column chromatography and obtains chloro-1 hydrogen of compound as white solid b:2--benzimidazole-4-methyl formate (1.5g, yield 100%).MS(ESI)m/z:[M+H]+=211。
The preparation of step 3:2-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate
Will dissolved with chloro-1 hydrogen of compound b:2--benzimidazole-4-methyl formate (59mg, dimethylformamide (5mL) 0.28mmol) adds piperazine (110mg, 1.12mmol), it is warming up to 100 DEG C, cool down after reacting 8 hours, removal of solvent under reduced pressure, residue separates (dichloromethane: methanol=10:1) through rapid column chromatography and obtains compound as white solid c:2-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (100mg, yield 100%).MS(ESI)m/z:[M+H]+=261。
The preparation of step 4:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Will dissolved with compound c:2-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (100mg, tetrahydrofuran solution (5mL) 0.28mmol) adds ammonia (5mL), it is warming up to 70 DEG C, tube sealing reaction cooled down after 8 hours, removal of solvent under reduced pressure, residue separates (dichloromethane: methanol=10:1) through rapid column chromatography and obtains compound as white solid d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (20mg, yield 28%).MS(ESI)m/z:[M+H]+=246。
The preparation of step 5:2-(4-(pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Will dissolved with compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (74mg, dimethylformamide (5mL) 0.3mmol) adds 2-chloropyrimide (34mg, 0.3mmol) with triethylamine (30mg, 0.3mmol), it is warming up to 100 DEG C, cool down after reacting 8 hours, removal of solvent under reduced pressure, residue separates (dichloromethane: methanol=10:1) through rapid column chromatography and obtains compound (1): 2-(4-(pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (32mg, yield 33%).LC-MS(ESI):m/z324(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.10(br,1H),9.16(br,1H),8.44-8.38(m,2H),7.62-7.54(m,2H),7.36-7.32(m,1H),7.01-6.95(m,1H),6.70-6.63(m,1H),3.89(br,4H),3.67(br,4H)。
Embodiment 2
The preparation of compound (2): 2-(4-(5-FU-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (2): 2-(4-(5-FU-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (30mg, yield 72%) is prepared with the chloro-5-FU generation aromatic nucleophilic substitution reaction of 2-by compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z342(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.92(br,1H),9.13(br,1H),8.50(s,2H),7.60(d,1H,J=7.8Hz),7.52(br,1H),7.32(d,1H,J=7.8Hz),6.98(t,1H,J=7.8Hz),3.87-3.83(m,4H),3.67-3.64(m,4H)。
Embodiment 3
The preparation of compound (3): 2-(4-(5-ethylamino-pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (3): 2-(4-(5-ethylamino-pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (23mg, yield 42%) is prepared with 2-chloro-5-ethylamino-pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z367(M+1)+.1HNMR(300MHz,DMSO-d6):δ9.08(br,1H),7.92(s,2H),7.78-7.72(m,2H),7.66-7.60(m,2H),7.22-7.16(m,1H),4.71-4.67(m,2H),4.19-4.15(m,2H),3.73-3.70(m,4H),2.65-2.60(m,2H),1.37(t,3H,J=4.5Hz)。
Embodiment 4
The preparation of compound (4): 2-(4-(5-acetamido pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (4): 2-(4-(5-acetamido pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (12mg, yield 22%) is prepared with 2-chloro-5-acetamido pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z381(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.85(br,1H),9.89(br,1H),9.14(s,1H),8.54(s,2H),7.60(d,1H,J=7.5Hz),7.51(br,1H),7.31(d,1H,J=7.5Hz),6.98(t,1H,J=7.5Hz),3.84-3.65(m,8H),2.00(s,3H)。
Embodiment 5
The preparation of compound (5): 2-(4-(5-methoxy pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (5): 2-(4-(5-methoxy pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (17mg, yield 41%) is prepared with 2-chloro-5-methoxyl pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z354(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.86(br,1H),9.15(br,1H),8.25(s,2H),7.60(d,1H,J=7.5Hz),7.51(br,1H),7.31(d,1H,J=7.5Hz),6.98(t,1H,J=7.5Hz),3.77(br,7H),3.64(br,4H)。
Embodiment 6
The preparation of compound (6): 2-(4-(5-amine pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (6): 2-(4-(5-amine pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (190mg, yield 83%) is prepared with 2-chloro-5-amine pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z339(M+1)+.1HNMR(300MHz,DMSO-d6):δ9.12(br,1H),7.60-7.25(m,7H),7.00-6.95(m,1H),3.67(br,8H)。
Embodiment 7
The preparation of compound (7): 2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (7): 2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (25mg, yield 65%) is prepared with 4-chloropyrimide generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z324(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.85(br,1H),9.09(br,1H),8.53(s,1H),8.22(d,1H,J=8.1Hz),7.60(d,1H,J=7.5Hz),7.50(br,1H),7.33(d,1H,J=7.5Hz),6.99(t,1H,J=7.5Hz),6.91(d,1H,J=8.1Hz),3.80-3.79(m,4H),3.68-3.66(m,4H)。
Embodiment 8
The preparation of compound (8): 2-(4-(3-ethylamino-pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (8): 2-(4-(3-ethylamino-pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (13mg, yield 36%) is prepared with 2-chloro-3-ethylamino-pyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z366(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.02(br,1H),9.18(br,1H),7.62-7.50(m,4H),7.34-7.31(m,1H),7.00-6.89(m,3H),3.77-3.74(m,4H),3.14-3.10(m,4H),2.00-1.93(m,2H),0.85-0.80(m,3H)。
Embodiment 9
The preparation of compound (9): 2-(4-(4-trifluoromethyl pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (9): 2-(4-(4-trifluoromethyl pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (36mg, yield 55%) is prepared with 2-chloro-4-trifluoromethyl pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z392(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.87(br,1H),9.13(br,1H),8.72(d,1H,J=4.8Hz),7.61(d,1H,J=7.8Hz),7.53(br,1H),7.33(d,1H,J=7.8Hz),7.07(d,1H,J=4.8Hz),6.99(t,1H,J=7.8Hz),3.94(br,4H),3.69(br,4H)。
Embodiment 10
The preparation of compound (10): 2-(4-(6-trifluoromethyl pyrimidine-4-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (10): 2-(4-(6-trifluoromethyl pyrimidine-4-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (40mg, yield 61%) is prepared with 6-chloro-4-trifluoromethyl pyrimidine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z392(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.88(br,1H),9.12(br,1H),8.66(s,1H),7.61(d,1H,J=7.5Hz),7.53(br,1H),7.35(s,1H),7.33(d,1H,J=7.5Hz),6.99(t,1H,J=7.5Hz),3.92(br,4H),3.69(br,4H)。
Embodiment 11
The preparation of compound (11): 2-(4-(5-methylcarbamoyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (11): 2-(4-(5-methylcarbamoyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (15mg, yield 24%) is prepared with 6-chloro-3-methylcarbamoyl pyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z380(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.86(br,1H),9.14(br,1H),8.6(s,1H),8.24(br,1H),7.96(d,1H,J=9.6Hz),7.61(d,1H,J=7.8Hz),7.52(br,1H),7.32(d,1H,J=7.8Hz),7.01-6.92(m,2H),3.77(br,4H),3.67(br,4H),2.74(d,3H,d=4.2Hz)。
Embodiment 12
The preparation of compound (12): 2-(4-(5-amine formyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (12): 2-(4-(5-amine formyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (25mg, yield 41%) is prepared with 6-chloro-3-amine formyl pyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z366(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.86(br,1H),9.14(br,1H),8.64(s,1H),7.99(d,1H,J=7.8Hz),7.79(br,1H),7.60(d,1H,J=9.0Hz),7.51(br,1H),7.32(d,1H,J=7.8Hz),7.17(br,1H),7.01-6.91(m,2H),3.78(br,4H),3.67(br,4H)。
Embodiment 13
The preparation of compound (13): 2-(4-(2-trifluoromethyl pyridine-4-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (13): 2-(4-(2-trifluoromethyl pyridine-4-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (7mg, yield 13%) is prepared with 4-chloro-2-trifluoromethyl pyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z391(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.88(br,1H),9.13(br,1H),8.34-8.29(m,1H),7.63-7.60(m,1H),7.53(br,1H),7.35-7.30(m,2H),7.12-7.09(m,1H),7.03-6.97(m,1H),3.70-3.64(m,8H)。
Embodiment 14
The preparation of compound (14): 2-(4-(5-cyanopyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (14): 2-(4-(5-cyanopyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (40mg, yield 71%) is prepared with 2-chloro-5-cyano group miaow pyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z349(M+1)+.1HNMR(300MHz,DMSO-d6):δ11.88(br,1H),9.09(br,1H),8.80(s,2H),7.60(d,1H,J=7.2Hz),7.53(br,1H),7.33(d,1H,J=7.2Hz),6.99(t,1H,J=7.2Hz),4.01(br,4H),3.69(br,4H)。
Embodiment 15
The preparation of compound (15): 2-(4-(5-dimethyl amine formylpyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (15): 2-(4-(5-dimethyl amine formylpyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (35mg, yield 44%) is prepared with 6-chloro-3-dimethyl amine formylpyridine generation aromatic nucleophilic substitution reaction by compound d:2-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z394(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.11(br,1H),9.19(br,1H),8.24(s,1H),7.66-7.59(m,2H),7.53(br,1H),7.34-7.31(m,1H),7.00-6.91(m,2H),3.73-3.70(m,8H),2.96(s,6H)。
Embodiment 16
Compound (16): the preparation of the fluoro-2-of 6-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide, concrete reaction equation is as follows:
Step 1:5-fluoro-3-nitro-2-(2,2,2-trifluoroacetamide) benzoic preparation:
Under ice bath, by fluoro-for 2-trifluoroacetamide-5-benzoic acid (2.5g, 10mmol) add in fuming nitric aicd (14mL) slowly, reaction is poured in frozen water after continuing stirring under ice bath 1 hour, it is filtrated to get the fluoro-3-nitro-2-(2 of compound as white solid e:5-, 2,2-trifluoroacetamides) benzoic acid (1.9g, yield 65%).MS(ESI)m/z:[M-H]-=295。
The preparation of the fluoro-3-nitrobenzoic acid of step 2:2-amido-5-:
Adding 10% sodium hydrate aqueous solution (20mL) dissolved with Verbindung: in the alcoholic solution (20mL) of the fluoro-3-nitro-2-(2,2,2-trifluoroacetamide) benzoic acid (1.18g, 4mmol) of 5-, reaction is warming up to 80 DEG C and stirs 3 hours.Decompression removes ethanol, residue salt acid for adjusting pH to 4, filters, obtains the yellow solid compound fluoro-3-nitrobenzoic acid of f:2-amido-5-(0.72g, yield 90%) MS (ESI) m/z:[M-H]-=199。
The preparation of the fluoro-3-nitrobenzene methyl of step 3:2-amido-5-:
Under ice bath, thionyl chloride (2.38g) is slowly added dropwise into dissolved with the fluoro-3-nitrobenzoic acid (0.8g of compound f:2-amido-5-, in methanol solution (20mL) 4mmol), heating is to refluxing, cool down after reacting 8 hours, removal of solvent under reduced pressure, residue separates (petroleum ether: ethyl acetate=5:1) through rapid column chromatography and obtains the yellow solid compound fluoro-3-nitrobenzene methyl of g:2-amido-5-(0.5g, yield 58%).MS(ESI)m/z:[M+H]+=215。
The preparation of step 4:2,3-bis-amido-5-fluorophenyl carbamate
10% palladium carbon (0.7g) is added dissolved with the fluoro-3-nitrobenzene methyl (7g of compound g:2-amido-5-, 32.7mmol) methanol (50mL) solution in, hydrogenate 7 hours under room temperature, filter, residue separates (petroleum ether: ethyl acetate=5:1) through rapid column chromatography and obtains yellow solid compound h:2,3-bis-amido-5-fluorophenyl carbamate (2.16g, yield 36%).MS(ESI)m/z:[M+H]+=185。
The preparation of step 5:6-fluoro-2-oxo-2,3-dihydro-1 hydrogen-benzimidazole-4-methyl formate
Embodiment 1 is adopted to prepare the method that compound (1) step 1 is similar,, by compound h:2,3-bis-amido-5-fluorophenyl carbamate and carbonyl dimidazoles (CDI) cyclization prepare the fluoro-2-oxo-2 of compound i:6-, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (711mg, yield 37%).MS(ESI)m/z:[M+H]+=211。
The preparation of fluoro-1 hydrogen of the chloro-6-of step 6:2--benzimidazole-4-methyl formate
Embodiment 1 is adopted to prepare the method that compound (1) step 2 is similar, by the fluoro-2-oxo-2 of compound i:6-, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate prepares compound j:2-fluoro-1 hydrogen of chloro-6--benzimidazole-4-methyl formate (681mg, yield 94%) with phosphorus oxychloride generation chlorination.MS(ESI)m/z:[M+H]+=229。
The preparation of the fluoro-2-of step 7:6-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate:
Embodiment 1 is adopted to prepare the method that compound (1) step 3 is similar, the fluoro-2-of compound k:6-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (430mg, yield 65%) is prepared with piperazine generation nucleophilic substitution by compound j:2-fluoro-1 hydrogen of chloro-6--benzimidazole-4-methyl formate.MS(ESI)m/z:[M+H]+=279。
The preparation of the fluoro-2-of step 8:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Will dissolved with the fluoro-2-of compound k:6-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (100mg, tetrahydrofuran solution (5mL) 0.28mmol) adds ammonia (5mL), it is warming up to 70 DEG C, tube sealing reaction cooled down after 8 hours, removal of solvent under reduced pressure, residue separates (dichloromethane: methanol=10:1) through rapid column chromatography and obtains the fluoro-2-of compound as white solid l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (20mg, yield 28%).MS(ESI)m/z:[M+H]+=246。
The preparation of the fluoro-2-of step 9:6-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (16) is prepared with 4-chloropyrimide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(pyrimidine-4-yl) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (21mg, yield 48%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z342(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.10(br,1H),8.52(s,1H),8.22(d,1H,J=7.2Hz),7.71(br,1H),7.33-7.2(m,1H),7.19-7.17(m,1H),6.90(d,1H,J=7.2Hz),3.80(br,4H),3.66(br,4H)。
Embodiment 17
Compound (17): the preparation of the fluoro-2-of 6-(4-(5-FU-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (17) is prepared with 2-chloro-5-FU generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-FU-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (27mg, yield 87%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z360(M+1)+.1HNMR(300MHz,DMSO-d6):δ9.11(br,1H),8.49(s,2H),7.71-7.69(m,1H),7.31-7.28(m,1H),7.18-7.15(m,1H),3.84-3.82(m,4H),3.68-3.65(m,4H)。
Embodiment 18
The preparation of compound (18): 2-(4-(5-(diformamide) pyridine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (18): 2-(4-(5-(diformamide) pyridine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (14mg, yield 18%) is prepared with 6-chloro-3-dimethyl amine formylpyridine generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z412(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.57(br,1H),9.12(br,1H),8.23(s,1H),7.70-7.63(m,2H),7.31-7.27(m,1H),7.18-7.14(m,1H),6.94-6.91(m,1H),3.72(br,8H),2.95(s,6H)。
Embodiment 19
The preparation of compound (19): 2-(4-(5-cyanopyrimidine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (19): 2-(4-(5-cyanopyrimidine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (43mg, yield 77%) is prepared with 2-chloro-5-cyano group miaow pyridine generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z367(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.11(br,1H),9.07(br,1H),8.80(s,2H),7.72(br,1H),7.33-7.29(m,1H),7.20-7.16(m,1H),4.00(br,4H),3.69(br,4H)。
Embodiment 20
Compound (20): the preparation of the fluoro-2-of 6-(4-(3-methyl nitrosourea pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (20) is prepared with 2-chloro-n-methyl nicotiamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(3-methyl nitrosourea pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (28mg, yield 52%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z398(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.21(br,1H),9.10(br,1H),8.41(br,1H),8.26-8.25(m,1H),7.75-7.72(m,1H),7.68(br,1H),7.32-7.28(m,1H),7.18-7.15(m,1H),6.96-6.92(m,1H),3.69(br,8H),2.79(s,3H)。
Embodiment 21
Compound (21): the preparation of the fluoro-2-of 6-(4-(5-trifluoromethyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (21) is prepared with 2-chloro-5-trifluoromethylpyridine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-trifluoromethyl pyridine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (23mg, yield 52%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z409(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.32(br,1H),9.10(br,1H),8.44(s,1H),7.85-7.81(m,1H),7.70(br,1H),7.32-7.28(m,1H),7.19-7.15(m,1H),7.06-7.02(m,1H),3.81(br,4H),3.70(br,4H).
Embodiment 22
Compound (22): the preparation of the fluoro-2-of 6-(4-(5-methyl nitrosourea yl pyrimidines-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (22) is prepared with 2-chloro-n-methyl pyrimidine-5-Methanamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-methyl nitrosourea yl pyrimidines-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (17mg, yield 29%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z399(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.01(br,1H),9.08(br,1H),8.78(s,2H),8.37(br,1H),7.73(br,1H),7.34-7.27(m,1H),7.19-7.13(m,1H),3.98(br,4H),3.67(br,4H),2.75(s,3H)。
Embodiment 23
Compound (23): the preparation of the fluoro-2-of 6-(4-(6-methyl nitrosourea radical pyridazine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (23) is prepared with 6-chloro-n-methyl pyridazine-3-Methanamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(6-methyl nitrosourea radical pyridazine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (20mg, yield 27%) LC-MS (ESI): m/z399 (M+1) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide+.1HNMR(300MHz,DMSO-d6):δ12.05(br,1H),9.11(br,1H),8.84(br,1H),7.87(d,1H,J=10.5Hz),7.74(br,1H),7.44-7.41(m,1H),7.31(d,1H,J=10.5Hz),7.20-7.17(m,1H),3.90(br,4H),3.72(br,4H),2.80(s,3H)。
Embodiment 24
Compound (24): the preparation of the fluoro-2-of 6-(4-(5-methyl nitrosourea yl pyridines-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide:
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (24) is prepared with 6-chloro-n-methyl nicotiamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-methyl nitrosourea yl pyridines-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (6mg, yield 13%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z398(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.58(br,1H),9.12(br,1H),8.62(s,1H),8.04-7.98(m,1H),7.72(s,1H),7.32-7.26(m,2H),7.17-7.15(m,1H),6.98-6.92(m,1H),3.75-3.69(m,8H),2.73(s,3H)。
Embodiment 25
Compound (25): the preparation of the fluoro-2-of 6-(4-(5-methyl nitrosourea base pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (25) is prepared with 5-chloro-n-methyl pyrazine-2-Methanamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-methyl nitrosourea base pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (38mg, yield 64%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z399(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.05(br,1H),9.09(br,1H),8.62(s,1H),8.39(br,1H),8.34(s,1H),7.72(br,1H),7.33-7.29(m,1H),7.19-7.17(m,1H),3.88(br,4H),3.70(br,4H),2.77(s,3H)。
Embodiment 26
The preparation of compound (26): 2-(4-(5-buserelin base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (26): 2-(4-(5-buserelin base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (19mg, yield 31%) is prepared with 5-chloro-N-ethyl pyrazine-2-Methanamide generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z413(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.09(br,1H),9.11(br,1H),8.63(s,1H),8.44-8.42(m,1H),8.34(s,1H),7.73(br,1H),7.33-7.29(m,1H),7.20-7.17(m,1H),3.87(br,4H),3.71(br,4H),3.28(q,2H,J=6.9Hz),1.09(t,3H,J=6.9Hz)。
Embodiment 27
Compound (27): the preparation of the fluoro-2-of 6-(4-(5-Isopropyl amide base pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (27) is prepared with 5-chloro-N-isopropylpyrazine-2-Methanamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-Isopropyl amide base pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (22mg, yield 28%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z427(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.09(br,1H),8.63(s,1H),8.32(s,1H),8.05-8.00(m,1H),7.70(br,1H),7.34-7.29(m,1H),7.20-7.16(m,1H),4.09(sep,1H,J=6.6Hz),3.88(br,4H),3.71(br,4H),1.15(d,6H,J=6.6Hz)。
Embodiment 28
The preparation of compound (28): 2-(4-(5-tert-butylamides base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (28): 2-(4-(5-tert-butylamides base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (29mg, yield 35%) is prepared with 5-chloro-N-tert-butyl-pyrazin-2-Methanamide generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z441(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.09(br,1H),8.62(s,1H),8.31(s,1H),7.71(br,1H),7.53(br,1H),7.33-7.29(m,1H),7.20-7.16(m,1H),3.86(br,4H),3.71(br,4H),1.37(s,9H)。
Embodiment 29
Compound (29): the preparation of the fluoro-2-of 6-(4-(5-(pyrrolin-1-acyl group) pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (29) is prepared with (5-chloropyrazine-2-base) (pyrrolin-1-base) ketone generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-(pyrrolin-1-acyl group) pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (14mg, yield 17%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z441(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.08(br,1H),8.51(s,1H),8.34(s,1H),7.71(br,1H),7.33-7.29(m,1H),7.19-7.16(m,1H),3.86(br,4H),3.70(br,4H),3.47-3.45(m,4H),1.86-1.83(m,4H)。
Embodiment 30
Compound (30): the preparation of the fluoro-2-of 6-(4-(5-(morpholine-4-acyl group) pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar; compound (30) is prepared with (5-chloropyrazine-2-base) (morpholine-2-base) ketone generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-(pyrrolin-1-acyl group) pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (32mg, yield 37%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z455(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.09(br,1H),8.41(s,1H),8.35(s,1H),7.71(br,1H),7.34-7.30(m,1H),7.20-7.16(m,1H),3.85(br,6H),3.70(br,4H),3.62(br,6H)。
Embodiment 31
Compound (31): the preparation of the fluoro-2-of 6-(4-(6-trifluoromethylpyridazine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (31) is prepared with 3-chlorin-6-trifluoromethylpyridazine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(6-trifluoromethylpyridazine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (15mg, yield 20%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z410(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.05(br,1H),9.10(br,1H),7.94-7.85(m,1H),7.71(br,1H),7.51-7.47(m,1H),7.34-7.30(m,1H),7.21-7.17(m,1H),3.93(br,4H),3.72(br,4H)。
Embodiment 32
Compound (32): the preparation of the fluoro-2-of 6-(4-(6-trifluoromethyl pyridine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (32) is prepared with 5-bromo-2-trifluoromethyl pyridine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(6-trifluoromethyl pyridine-3-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (23mg, yield 19%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z409(M+1)+.1HNMR(300MHz,DMSO-d6):δ9.09(br,1H),8.50(s,1H),7.72-7.66(m,2H),7.52-7.48(m,1H),7.33-7.28(m,1H),7.20-7.16(m,1H),3.72(br,4H),3.54(br,4H)。
Embodiment 33
Compound (33): the preparation of the fluoro-2-of 6-(4-(2-trifluoromethyl pyrimidine-5-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (33) is prepared with 5-bromo-2-trifluoromethyl pyrimidine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(2-trifluoromethyl pyrimidine-5-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (12mg, yield 16%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z410(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.09(br,1H),8.69(s,2H),7.71(br,1H),7.32-7.29(m,1H),7.20-7.17(m,1H),3.73(br,4H),3.62(br,4H)。
Embodiment 34
Compound (34): the preparation of the fluoro-2-of 6-(4-(5-trifluoromethyl pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (34) is prepared with 2-chloro-5-trifluoromethyl pyrimidine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-trifluoromethyl pyrimidine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (8mg, yield 14%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z410(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.02(br,1H),9.09(br,1H),8.75(s,2H),7.72(br,1H),7.33-7.29(m,1H),7.20-7.17(m,1H),4.00(br,4H),3.69(br,4H)。
Embodiment 35
Compound (35): the preparation of the fluoro-2-of 6-(4-(5-trifluoromethyl pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (35) is prepared with 2-chloro-5-trifluoromethyl pyrazine generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(5-trifluoromethyl pyrazine-2-base) piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (60mg, yield 90%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z410(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.04(br,1H),9.09(br,1H),8.51(s,1H),8.50(s,1H),7.71(br,1H),7.33-7.30(m,1H),7.20-7.17(m,1H),3.89(br,4H),3.71(br,4H)。
Embodiment 36
The preparation of compound (36): 2-(4-(5-dimethylformamide base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide and the chloro-N of 5-, N-dimethyl pyrazine-2-Methanamide generation aromatic nucleophilic substitution reaction prepares compound (36): 2-(4-(5-dimethylformamide base pyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (29mg, yield 37%).LC-MS(ESI):m/z413(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.02(br,1H),9.09(br,1H),8.37(s,1H),8.34(s,1H),7.71(br,1H),7.33-7.30(m,1H),7.20-7.16(m,1H),3.83(br,4H),3.73(br,1H),3.07(s,3H),2.98(s,3H)。
Embodiment 37
The preparation of compound (37): 2-(4-(5-cyanopyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (37): 2-(4-(5-cyanopyrazine-2-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (40mg, yield 58%) is prepared with 5-chloro-2 cyano pyrazine generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z367(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.05(br,1H),9.07(br,1H),8.59(s,1H),8.49(s,1H),7.69(s,1H),7.33-7.29(m,1H),7.20-7.16(m,1H),3.93(br,4H),3.71(br,4H)。
The preparation of compound (38): 2-(4-(2-cyanopyrimidine-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (38): 2-(4-(2-cyanopyrimidine-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (18mg, yield 26%) is prepared with 5-bromo-2-cyanopyrimidine generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z367(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.03(br,1H),9.08(br,1H),8.63(s,2H),7.73-7.65(m,1H),7.33-7.29(m,1H),7.22-7.17(m,1H),3.73-3.69(m,8H)。
Compound (39): the fluoro-2-of 6-(4-(2-methyl nitrosourea yl pyrimidines-2-base) piperazine-5-base)-1 hydrogen-h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (39) is prepared with 5-bromo-N-methylpyrimidine-2-Methanamide generation aromatic nucleophilic substitution reaction: the fluoro-2-of 6-(4-(2-methyl nitrosourea yl pyrimidines-2-base) piperazine-5-base)-1 hydrogen-h-benzimidazole-4-carboxamide (16mg, yield 29%) by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z399(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.02(br,1H),9.08(br,1H),8.67(s,2H),8.34(br,1H),7.71(br,1H),7.33-7.27(m,1H),7.19-7.12(m,1H),3.99(br,4H),3.68(br,4H),2.72(s,3H)。
The preparation of compound (40): 2-(4-(2-buserelin yl pyrimidines-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, compound (40): 2-(4-(2-buserelin yl pyrimidines-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide (17mg, yield 23%) is prepared with 5-bromo-N-ethyl-pyrimidine-2-Methanamide generation aromatic nucleophilic substitution reaction by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide.LC-MS(ESI):m/z413(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.01(br,1H),9.09(br,1H),8.64(s,2H),8.31(br,1H),7.72(br,1H),7.34-7.27(m,1H),7.19-7.13(m,1H),3.96(br,4H),3.65(br,4H),3.26(q,2H,J=6.9Hz),1.07(t,3H,J=6.9Hz)。
The preparation of compound (41): 2-(4-(2-dimethylformamide yl pyrimidines-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide
Embodiment 1 is adopted to prepare the method that compound (1) step 5 is similar, the preparation (19mg, yield 26%) of compound (41): 2-(4-(2-dimethylformamide yl pyrimidines-5-base) piperazine-1-base) fluoro-1 hydrogen of-6--h-benzimidazole-4-carboxamide is prepared by the fluoro-2-of compound l:6-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide and 5-bromo-N-dimethyl pyrimidine-2-Methanamide generation aromatic nucleophilic substitution reaction.LC-MS(ESI):m/z413(M+1)+.1HNMR(300MHz,DMSO-d6):δ12.03(br,1H),9.07(br,1H),8.63(s,2H),8.32(br,1H),7.72(br,1H),7.31-7.26(m,1H),7.18-7.13(m,1H),3.97(br,4H),3.67(br,4H),3.08(s,3H),2.97(s,3H)。
Biological assessment
Experimental principle:
After the translation that the poly ADP of nucleoprotein is ribosylating when being to occur at DNA damage response.PARP, full name is Poly adenosine diphosphate-ribose polymerase-1, and when there being NAD to exist, catalysis poly (ADP-ribose) is connected on the nucleoprotein closed on, thus causing the DNA repair mechanism via base excision repair path.The HTUniversalChemiluminescentPARPAssayKit that Trevigen company produces can measure this by biotin labeled ADP-ribose and histone in conjunction with level.
Reagent and consumptive material
1.HTUniversalChemiluminescentPARPAssayKitwithHistone-coa tedStripWells, U.S. Trevigen, article No.: 4676-096-K.
2. read plate instrument, the U.S. PerkinElmer, EnVisionMultilabelPlateReader.
Solution and buffer
1. washing liquid contains the PBS solution of 0.1%TritonX-100.
20XPARP buffer is diluted 20 times and namely obtains 1X buffer by 2.20XPARP buffer deionized water, and this buffer is used to dilution restructuring PARP enzyme, PARPCocktail and tested compound.
3.10XPARPCocktail prepares 1XPARPCocktail:10XPARPCocktail2.5 μ l/well, 10X activated dna 2.5 μ l/well, 1XPARP buffer 20 μ l/well in accordance with the following methods.
4.PARPEnzyme only before use, carefully dilutes recombinase with 1XPARP buffer, and the enzymatic solution diluted to use as early as possible, unspent to discard.
5.Strep-HRP only before use, obtains 1X solution with 1XStrep diluted Strep-HRP500 times.
6. chemical luminous substrate is only before use, and the PeroxyGlowA of same volume and B solution mix homogeneously obtain the substrate of horseradish peroxidase.
Experimental technique
Compound is prepared
1. with DMSO, each for 10mM test compound mother solution is diluted to 10uM, 1uM.
2. only before experiment starts, the gradient concentration solution 1XPARP buffer of each compound being dissolved in DMSO dilutes 20 times, obtain the compound solution of 5X, namely can be utilized for detection, positive control (POSITIVE) and negative control (NEGATIVE) hole are 1XPARP buffer (DMSO content 5%).
Operating procedure
1. every hole adds 50 μ l1XPARP buffer moistening histones, incubated at room orifice plate 30 minutes, then by the 1XPARP buffer sucking-off in hole, and is patted dry only by residual liquid on napkin.
2. according to compound (1) to (37), being added in corresponding hole by the 5X compound solution diluted, every hole 10 μ l, positive control (POSITIVE) and negative control (NEGATIVE) hole are 1XPARP buffer (DMSO content 5%)
3. with 1XPARP buffer, PARP enzyme being diluted to every 15 μ l solution and contain 0.5Unit, then add 15 μ l enzymatic solution in other holes except negative control hole, negative control hole only adds 1XPARP buffer, incubated at room orifice plate 10 minutes.
4. continuously add the 1XPARPCocktail of 25 μ l in each hole.
Hatch orifice plate 60 minutes for 5.27 DEG C.
6., after hatching end, by the reactant liquor sucking-off in hole, and on napkin, residual liquid is patted dry only.Rinsing orifice plate 4 times followed by the PBS solution containing 0.1%TritonX-100, every hole is with 200 μ l every time, and is patted dry only by residual liquid on napkin.
7., it follows that add the 1XStrep-HRP solution diluted in every hole, then hatch orifice plate 60 minutes at 27 DEG C.
8., after hatching end, by the reactant liquor sucking-off in hole, and on napkin, residual liquid is patted dry only.Rinsing orifice plate 4 times followed by the PBS solution containing 0.1%TritonX-100, every hole is with 200 μ l every time, and is patted dry only by residual liquid on napkin.
9. after washing the bundle that hardens, by the PeroxyGlowA of same volume and B solution mix homogeneously, every hole adds 100 μ l, is immediately placed in reading plate instrument record chemiluminescence signal.
Data process
Reading in every hole needs to be converted into suppression ratio.The suppression ratio of compound can use following equation to calculate:
Note: Positive control wells reading is positive hole reading, meaning is enzyme 100% activity；Negative control hole reading is negative hole reading, and meaning is enzyme 0%；Active X is the reading of each each concentration of sample.
Table 1 compound inhibitory activity to PARP-1 enzyme
The activity data listed in table 1 fully shows, the compound of the present invention is all the inhibitor of PARP-1, wherein in embodiment, compound (1), (2), (5), (6), (7), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41) IC50Value is not more than 100nM, compound (16), (19), (25), (26), (30), (31), (33), (35), (36), (37), (38), (39), (40), the IC of (41)50Value is not more than 10nM.

Claims (13)

1. the compound shown in formula (33):
2. the compound shown in formula (33) as claimed in claim 1, it is characterised in that described formula (33) compound exists as a pharmaceutically acceptable salt form.
3. the compound shown in formula (33) as claimed in claim 2, it is characterized in that, described pharmaceutically acceptable salt is the hydrochlorate of formula (33) compound, sulfate, phosphate, acetate, trifluoroacetate, mesylate, fluoroform sulphonate, tosilate, tartrate, maleate, fumarate, succinate or malate.
4. the preparation method of the compound shown in formula described in claim 1 (33), its reaction equation is as follows:
Specifically comprise the following steps that
Step 1): the fluoro-2-amido benzoic acid of 5-and trifluoroacetic acid anhydride reactant, obtain the 5-fluoro-2-amido benzoic acid (II) of trifluoroacetyl group protection；
Step 2): the fluoro-2 amido benzoic acid (II) of 5-of trifluoroacetyl group protection react with fuming nitric aicd, obtain the 5-fluoro-2-amido-3 nitrobenzoic acid (III) of trifluoroacetyl group protection；
Step 3): the 5-fluoro-2-amido-3 nitrobenzoic acid (III) of trifluoroacetyl group protection reacts in methanol with thionyl chloride, obtains 5-fluoro-2-amido-3 nitrobenzene methyl (IV)；
Step 4): 5-fluoro-2-amido-3 nitrobenzene methyl (IV) uses palladium carbon catalytic hydrogenation, obtains fluoro-2,3-diamidogen yl benzoic acid methyl ester (V) of 5-；
Step 5): fluoro-2,3-diamidogen yl benzoic acid methyl ester (V) of 5-and carbonyl dimidazoles ring-closure reaction, obtain fluoro-2-oxo-2 of 5-, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (VI)；
Step 6): fluoro-2-oxo-2 of 5-, 3-dihydro-1 hydrogen-benzimidazole-4-methyl formate (VI) and phosphorus oxychloride carry out chlorination reaction, obtain 5-chloro-1 hydrogen of fluoro-2--benzimidazole-4-methyl formate (VII)；
Step 7): chloro-1 hydrogen of the fluoro-2-of 5--benzimidazole-4-methyl formate (VII) and piperazine carry out nucleophilic substitution, obtain the fluoro-2-of 5-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (VIII)；
Step 8): there is the aminolysis reaction of ester group in the fluoro-2-of 5-(piperazine-1-base)-1 hydrogen-benzimidazole-4-methyl formate (VIII), obtain the fluoro-2-of 5-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (Ⅸ) in methanolic ammonia solution；
Step 9): the fluoro-2-of 5-(piperazine-1-base)-1 hydrogen-h-benzimidazole-4-carboxamide (Ⅸ) and 5-chloro-2-cyanopyrimidine generation aromatic nucleophilic substitution reaction, obtain the compound shown in formula (33).
5. the application in the disease medicament that preparation treatment improves because PARP activity suppresses of formula (33) compound described in claim 1 or 2 or 3.
6. apply as claimed in claim 5, it is characterised in that the described disease improved because PARP activity suppresses is angiopathy, septic shock, ischemic injuries, neurotoxicity, hemorrhagic shock, inflammatory diseases or multiple sclerosis.
7. the pharmaceutically useful salt of formula described in Claims 2 or 3 (33) compound application in the disease medicament that preparation treatment improves because PARP activity suppresses.
8. applying as claimed in claim 7, the described disease improved because PARP activity suppresses is angiopathy, septic shock, ischemic injuries, neurotoxicity, hemorrhagic shock, inflammatory diseases or multiple sclerosis.
9. formula (33) compound described in claim 1 or 2 or 3 is used for the application in the ancillary drug of oncotherapy in preparation.
10. formula (33) compound described in claim 1 or 2 or 3 lacks the application in the medicine of the individuation treatment of cancer that homologous recombination (HR) dependent DNA double chain interruption (DSB) is repaired in preparation.
11. apply as claimed in claim 10, wherein said cancer is the cancer of the cancerous cell being lowered relative to normal cell by the ability of the DSB of HR DNA plerosis containing one or more or losing.
12. apply as claimed in claim 11, wherein said cancer is the cancer with BRCA-1 or BRCA-2 defect, mutant phenotype.
13. apply as claimed in claim 12, described cancer is breast carcinoma, ovarian cancer, cancer of pancreas or carcinoma of prostate.
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CN1332731A (en) * 1998-11-27 2002-01-23 巴斯福股份公司 Substituted benzimidazoles and their prep. and use
CN101155797A (en) * 2005-04-11 2008-04-02 艾博特公司 1h-benzimidazole-4-carboxamides substituted with a quaternary carbon at the 2-position are potent parp inhibitors

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