CN102134243A - Bicyclic amino pyrazole derivatives with anti-tumor activity - Google Patents

Bicyclic amino pyrazole derivatives with anti-tumor activity Download PDF

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CN102134243A
CN102134243A CN2011100520021A CN201110052002A CN102134243A CN 102134243 A CN102134243 A CN 102134243A CN 2011100520021 A CN2011100520021 A CN 2011100520021A CN 201110052002 A CN201110052002 A CN 201110052002A CN 102134243 A CN102134243 A CN 102134243A
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pyrazole
phenyl
methane amide
pyrrolin
benzoyl
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王玉成
白晓光
邵荣光
王菊仙
何红伟
张�浩
刘洪涛
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Institute of Medicinal Biotechnology of CAMS
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The invention relates to bicyclic amino pyrazole derivatives and an application thereof in treatment of tumors, research results show that compounds have a role in significantly inhibiting tumor cells, wherein a plurality of compounds are obviously better than a positive control medicament, namely Rescovitine, on the cell level, and the bicyclic amino pyrazole derivatives are expected to be studied and developed into clinically effective anti-tumor medicaments.

Description

Bicyclic amino group pyrazole derivatives with anti-tumor activity
Technical field:
The present invention relates to have anti-tumor activity, the active dicyclo pyrazole derivatives of solid tumor resisting particularly.
Background technology:
Amino pyrazoles compounds is many important skeletons with active compound for anti tumor, and at present known numerous clinical antitumor candidate compounds preceding and clinical study all have the structure of amino-pyrazol.United States Patent (USP) successively discloses a series of aminopyrazole compounds (US6218418, US20030171357A1) that can be used as kinases inhibitor.Discover that this compounds optionally suppresses some kinases relevant with tumour, as Aurora-A, CDK2/cyclinA etc., and then the highly selective high efficiency suppresses the growth of various human tumor cell line.Amino pyrazoles compounds is the bicyclic amino group pyrazole compound especially, all shows excellent pharmacologically active in antitumor or the unusual relevant oncotherapy of kinases, thereby has the antitumor exploitation value of potential.
Bicyclic amino group pyrazole compound of the present invention and in the application of anti-tumor aspect, not seeing as yet so far has relevant report both domestic and external.
One of purpose of the present invention is that the pyrazole derivatives of the bicyclic amino group with antitumor activity is provided.
Two of purpose of the present invention is that the preparation method of bicyclic amino group pyrazole derivatives is provided.
Three of purpose of the present invention is that the antineoplastic pharmaceutical compositions that contains the bicyclic amino group pyrazole derivatives is provided.
Four of purpose of the present invention is that the antitumor application of described derivative and composition thereof is provided.
Summary of the invention:
The structure of bicyclic amino group pyrazole derivatives provided by the present invention is suc as formula shown in (I):
Figure BSA00000444100100011
In the formula:
R 1Independently be selected from: C 1-C 10Alkyl, C 2-C 10Alkenyl, C 2-C 10Alkynyl group, C 3-C 8Cycloalkyl, C 4-C 12Cycloalkylalkyl, aryl, heteroaryl ,-COR 4,-COOR 4,-CONHR 4,-CONR 4R 5,-C (=NH) NHR 4,-SO 2R 4,-SO 2NHR 4,-SO 2NR 4R 5
R 2, R 3The independent hydrogen atom or optional of representing from R 4,-COR 4,-COOR 4,-CONHR 4,-CONR 4R 5,-C (=NH) NHR 4,-SO 2R 4,-SO 2NHR 4,-SO 2NR 4R 5
M and n represent 1,2 separately;
C 1-C 10Alkyl, C 2-C 10Alkenyl, C 2-C 10Alkynyl group, C 3-C 8Cycloalkyl, C 4-C 12Cycloalkylalkyl is optional independently to be selected from following substituting group replacement: C by 1-3 1-C 4Alkyl, nitro, halogen, cyano group, NR 4R 5, NR 4COR 5, NR 4CO 2R 5, COR 4, OR 4, CONR 4R 5, CO 2R 4
R 4And R 5Independently be selected from hydrogen atom or C 1-C 6Alkyl, C 3-C 6Cycloalkyl, C 4-C 6Alkenyl, C 4-C 6Alkynyl group, aryl, heteroaryl;
Aryl is a phenyl or naphthyl, and each is chosen wantonly by 1-5 and independently is selected from following substituting group replacement: C 1-C 6Alkyl, C 3-C 6Cycloalkyl, halogen, C 1-C 4Haloalkyl, cyano group, nitro ,-NR 6R 7,-CONR 6R 7,-NR 6COR 8,-OR 9,-COOR 9,-SR 9,-SO 2R 9,-SO 2NR 6R 7,-NR 6SO 2R 9
Heteroaryl is pyridyl, pyrimidyl, triazinyl, furyl, pyranyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indyl, pyrryl, oxazolyl, benzofuryl, benzothiazolyl, isoxazolyl, pyrazolyl, 2,3-dihydrobenzo thienyl or 2, the 3-dihydro benzo furyl, each is chosen wantonly by 1～5 and independently is selected from following substituting group replacement: C 1-C 6Alkyl, C 3-C 6Cycloalkyl, halogen, C 1-C 4Haloalkyl, cyano group, nitro ,-NR 6R 7,-CONR 6R 7,-NR 9COR 8,-OR 9,-COOR 9,-SR 9,-SO 2R 9,-SO 2NR 6R 7,-NR 6SO 2R 9
R 6, R 7, R 8, R 9Independent separately hydrogen atom or the C of representing 1-C 6Alkyl or phenyl.
The compound of general formula (I) exists with geometry and optical isomer, and all isomer are isolating, pure or partially purified steric isomer or its racemic mixture.
The compound of general formula (I) is optionally with pharmaceutically-acceptable acid addition, metal-salt or selective alkylation ammonium salt exist, comprise inorganic and organic acid salt, such as hydrochloride, hydrobromate, vitriol, phosphoric acid salt, acetate, trifluoroacetate, succinate, fumarate, maleate, Citrate trianion, lactic acid salt, tartrate, mandelate, oxalate or similar pharmaceutically acceptable inorganic or organic acid addition salt, also their an alkali metal salt of conversion processes routinely, alkaline earth salt, silver salt, barium salt etc.
Compound of the present invention can be used to cause the agent combination administration of tumour regression with other, to coordinate to increase the antitumous effect of described compound.The compound that can be used for uniting use with the present invention be selected from cis-platinum, carboplatin, Zorubicin, holder pool for may, taxol, taxotere, vincristine(VCR) and 5 FU 5 fluorouracil etc.
Pharmaceutical composition of the present invention comprises the compound at least a of the present invention for the treatment of effective dose and the mixture of pharmaceutically useful vehicle.
Oral compositions comprises inert diluent or edible carrier usually, can be contained in the gelatine capsule or is compressed into tablet.Other is applicable to that the form of oral administration is capsule, pill, suspension or syrup.
Tablet, pill, capsule or similar compositions can comprise following compositions (except that active substance): tackiness agent such as Microcrystalline Cellulose, tragacanth or gelatin; Vehicle such as starch or lactose; Disintegrating agent such as alginic acid, former gel (Primogel), W-Gum etc.; Lubricant such as Magnesium Stearate; Antiseize paste such as colloidal silica; Sweeting agent such as sucrose or asccharin; Correctives such as peppermint, wintergreen oil or orange fragrance matter.When the composition of selecting was capsule, it can also comprise liquid vehicle such as fatty oil.Other composition can comprise other the various physical form materials that improve, as Drug coating (to tablet and pill) sugar or shellac.The material that is used to prepare composition should be pharmaceutically pure, and is nontoxic under the dosage that is adopted.
For preparation is used for the pharmaceutical composition of administered parenterally, activeconstituents can be mixed in solution or the suspension, it also comprises following component: sterile diluent such as water for injection, salt brine solution, oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antiseptic-germicide such as benzyl alcohol; Antioxidant such as xitix or sodium bisulfite; Sequestrant such as ethylenediamine tetraacetic acid (EDTA); Buffer reagent such as acetate, Citrate trianion or phosphoric acid salt are used for regulator solution tensile reagent such as sodium-chlor or glucose.Parenteral administration can be contained in ampoule, disposable syringe or glass or the plastics phial.
The present invention specifically comprises following compound, and the mixture of their isomer, steric isomer or steric isomer and pharmacy acceptable salt or prodrug:
1) N-{5-benzoyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
2) N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
3) N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
4) N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
5) N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
6) N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
7) N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
8) N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
9) N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
10) N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
11) N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
12) N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
13) N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
14) N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
15) N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
16) N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
17) N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
18) N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
19) N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
20) N-{5-(4-methyl-benzoyl)-1-(4-methyl-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
21) N-{5-(4-methoxyl group-benzoyl)-1-(4-methoxyl group-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
22) N-{5-(4-fluoro-benzoyl)-1-(4-fluoro-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
23) N-{5-(1-benzyl)-1-(1-benzyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
24) N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
25) N-{5-(4-methyl-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
26) N-{5-(4-fluoro-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
27) N-{5-(4-methoxyl group-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
28) N-{5-(1-benzyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
29) N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
30) N-{5-(4-methyl-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
31) N-{5-(4-fluoro-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
32) N-{5-(4-methoxyl group-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
33) N-{5-(1-benzyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
34) N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
35) N-{5-(4-methyl-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
36) N-{5-(4-fluoro-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
37) N-{5-(4-methoxyl group-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
38) N-{5-(1-benzyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
39) N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
40) N-{5-(4-methyl-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
41) N-{5-(4-fluoro-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
42) N-{5-(4-methoxyl group-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
43) N-{5-(1-benzyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
Shown in the following route of preparation method of bicyclic amino group pyrazole compound of the present invention:
Figure BSA00000444100100061
Concrete steps are as follows:
1) starting raw material II can the reference similar approach be prepared (Gadekar, Sheekrishna M.et al.J Med.Chem.1968,11 (3), 616-618; Hong CY, et al.J Med.Chem.1997,40 (22), 3584-3593; Pedersen H et al.Bioorg.Med.Chem.1997,7,795-809), reference (Mikhail Krasavin﹠amp; Igor O.Konstantinov, Letters in Organic Chemistry, 2008,5,594-598) similar approach, II and acethydrazide are after reacting 2～24 hours under 0 ℃～100 ℃ in propyl carbinol, again with N methyl piperazine back flow reaction 8～24 hours, intermediate III.
2) reference (Daniele Fancelli, et al.J.Med.Chem.2005,48,3080-3084) similar approach, acid acceptor is arranged (as triethylamine, N, N-diisopropylethylamine, pyridine) exist down III and Vinyl chloroformate in polar aprotic solvent (as tetrahydrofuran (THF), methylene dichloride, acetonitrile, chloroform), reacted 0.5～24 hour down at-10 ℃～30 ℃, obtain intermediate compound IV.
3) reference (Daniele Fancelli; et al.J.Med.Chem.2005; 48; 3080-3084) similar approach; acid acceptor is arranged (as triethylamine, N; N-diisopropylethylamine, pyridine) exist down IV in polar aprotic solvent (as tetrahydrofuran (THF), methylene dichloride, acetonitrile, chloroform) ,-10 ℃ to reflux temperature, can obtain intermediate V with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively.
4) reference (Daniele Fancelli, et al.J.Med.Chem.2005,48,3080-3084) similar approach is reacted V 0.5～24 hour to reflux temperature in-10 ℃ in 10% triethylamine methanol solution, obtains intermediate VI.
5) acid acceptor is arranged (as triethylamine, N; N-diisopropylethylamine, pyridine) exist to descend with VI in polar aprotic solvent (as tetrahydrofuran (THF), methylene dichloride, acetonitrile, chloroform);-10 ℃ to reflux temperature, can obtain intermediate VII with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively.
6) reference (Bioorganic ﹠amp; Medicinal Chemistry Letters 16 (2006) 1084-1090) similar approach in anhydrous organic solvent, was reacted VII 1～24 hour under acidic conditions, obtain intermediate VIII.
7) acid acceptor is arranged (as triethylamine, N; N-diisopropylethylamine, pyridine) exist to descend with VIII in polar aprotic solvent;-10 ℃ to reflux temperature, can obtain formula (I) compound with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively.
The present invention also provides described compound and the application of composition in the preparation antitumor drug thereof.
Embodiment:
Following examples are only understood the present invention better for help those skilled in the art, but do not limit the present invention in any way.
＜embodiment 1〉intermediate 3-amino-4,6-dihydro-1H-pyrroles [3,4-c] pyrazoles-5-carboxylic acid tertiary butyl ester
Can be prepared (Mikhail Krasavin﹠amp with reference to the known references method; Igor O.Konstantinov, Letters in Organic Chemistry, 2008,5,594-598), detailed process is as follows: (synthetic method is referring to Daniele Fancelli, et al.J.Med.Chem.2005,48 for 1-tertbutyloxycarbonyl-3-cyano group-4-pyrrolidone, 3080-3084) 120.0g, 0.571mol be suspended in the 700mL propyl carbinol, add acethydrazide 50.8g, 0.685mol under the high degree of agitation, finish, being warming up to 70 ℃ and holding temperature gradually stirs and spends the night disposable adding N methyl piperazine 74.4g, 0.743mol, be heated to and reflux and reacted 12 hours, TLC thin plate chromatography is followed the tracks of reaction and is finished, and naturally cools to room temperature, adds isopyknic water and high degree of agitation 20-30min, standing demix, discard lower aqueous layer, the upper strata repeats to wash 2-4 time, the organic layer vacuum rotary steam to do the brown ceramic powder crude product, adding ethyl acetate 300mL grinds, dry behind the suction filtration white powder 102g, yield 79.7%, m.p.167-169 ℃.
1H?NMR(400MHz，DMSO-d 6)δppm：1.42(9H，s)，4.12(4H，m)，5.03(2H，broad?s)，11.18(1H，broad?s)。
MS(ESI，m/z)：225(M ++1，100％)
＜embodiment 2〉intermediate 1-ethoxycarbonyl-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles
Can be prepared (Daniele Fancelli with reference to the known references method, et al.J.Med.Chem.2005,48,3080-3084), detailed process is as follows: get Vinyl chloroformate (8.9mL, 93mmol) in 250mL tetrahydrofuran (THF) (reflux to lay equal stress on steam obtain through hydrolith), under 0-5 ℃, slowly drop to 3-amino-4, ((20g is 89mmol) with DIEA (92mL for embodiment 1 step a) for 6-dihydro-1H-pyrroles [3,4-c] pyrazoles-5-carboxylic acid tertiary butyl ester, in 500mL tetrahydrofuran (THF) 528mmol) (reflux to lay equal stress on to steam obtain through the hydrolith) solution, keep 0-5 ℃ 2 hours, allow to rise to room temperature, stirring is spent the night.The gained mixture is spin-dried for, and resistates is washed with ethyl acetate (200mL) dissolving, the organic layer anhydrous sodium sulfate drying, suction filtration is spin-dried for, resistates is through silicagel column separation and purification (petrol ether/ethyl acetate=10: 1), and vacuum-drying gets white powder solid 18g, yield 68%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.26-1.27(3H，2t)，1.44(9H，s)，4.14-4.20(2H，m)，4.26(2H，q)，4.44-4.47(2H，m)，5.69(2H，s)。
MS(ESI，m/z)：297(M ++1，100％)
＜embodiment 3〉intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
Can be prepared (Daniele Fancelli with reference to the known references method, et al.J.Med.Chem.2005,48,3080-3084), detailed process is as follows: commercially available Benzoyl chloride (5.6mL, 48mmol) in 50mL tetrahydrofuran (THF) (reflux to lay equal stress on steam obtain through hydrolith), under 0-5 ℃, slowly drop to embodiment 2 intermediate 1-ethoxycarbonyls-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles (13g, 44mmol) with the 500mL tetrahydrofuran (THF) of DIEA (47.4mL) (reflux to lay equal stress on steam obtain through hydrolith) solution in, keep 0-5 ℃ 2 hours, allow to rise to room temperature, stirring is spent the night.The gained mixture is spin-dried for, and resistates dissolves with ethyl acetate (600mL), saturated NaHCO 3Washing (300mL * 2) has a large amount of white solids to separate out in organic phase during washing, leave standstill, and leaches solid, gets white powder solid 10.9g, yield 62% after the drying.
1H?NMR(400MHz，DMSO-d 6)δppm：1.31-1.32(3H，2t，)，1.45(9H，s)，4.39(2H，q，)，4.50-4.52(2H，m)，4.57-4.59(2H，m)，7.53(3H，m)，8.03(2H，d)，11.48(1H，broads)。
MS(ESI，m/z)：401(M ++1，100％)
＜embodiment 4〉intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 3 described methods, with commercially available Butyltriphenylphosphonium chloride and embodiment 2 intermediate 1-ethoxycarbonyls-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles prepares title compound, the pulverulent solids that is white in color, yield 72%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.32-1.34(3H，2t)，1.45(9H，s)，2.36(3H，s)，4.37(2H，q)，4.49-4.52(2H，m)，4.57-4.60(2H，m)，7.28(2H，d)，7.93(2H，d，)，11.39(1H，broad?s)。
MS(ESI，m/z)：415(M ++1，100％)
＜embodiment 5〉intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 3 described methods, to fluorobenzoyl chloride and embodiment 2 intermediate 1-ethoxycarbonyls-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles prepares title compound, the pulverulent solids that is white in color, yield 75% with commercially available.
1H?NMR(400MHz，DMSO-d 6)δppm：1.31-1.34(3H，2t)，1.45(9H，s)，4.38(2H，q)，4.49-4.52(2H，m)，4.57-4.60(2H，m)，7.33(2H，t)，8.11(2H，m)，11.52(1H，broads)。
MS(ESI，m/z)：419(M ++1，100％)
＜embodiment 6〉intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 3 described methods, with commercially available anisoyl chloride and embodiment 2 intermediate 1-ethoxycarbonyls-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles prepares title compound, the pulverulent solids that is white in color, yield 70%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.30-1.34(3H，2t)，1.45(9H，s)，3.82(3H，s)，4.37(2H，q，J＝7.2Hz)，4.48-4.52(2H，m)，4.56-4.59(2H，m)，7.00(2H，d)，8.03(2H，d)，11.31(1H，broad?s)。
MS(ESI，m/z)：431(M ++1，100％)
＜embodiment 7〉intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
Phenyllacetyl chloride (7.36mL, 55mmol) in 50mL tetrahydrofuran (THF) (reflux to lay equal stress on steam obtain through hydrolith), under 0-5 ℃, slowly drop to embodiment 2 intermediate 1-ethoxycarbonyls-5-tertbutyloxycarbonyl-3-amino-4,6-pyrrolin [3,4-c] pyrazoles (15g, 50.6mmol) with the 400mL tetrahydrofuran (THF) of DIEA (50mL) (reflux to lay equal stress on steam obtain through hydrolith) solution in, keep 0-5 ℃ 2 hours, allow to rise to room temperature, stirring is spent the night.The gained mixture is spin-dried for, and resistates dissolves with ethyl acetate (500mL), saturated NaHCO 3Washing (200mL * 4), the organic layer anhydrous sodium sulfate drying, suction filtration, filtrate decompression is removed and is desolvated, and resistates is through silicagel column separation and purification (petrol ether/ethyl acetate=10: 1), and vacuum-drying gets white powder solid 14.3g, yield 68%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.30-1.34(3H，2t)，1.45(9H，s)，3.82(3H，s)，4.37(2H，q)，4.51-4.53(2H，m)，4.56-4.59(2H，m)，7.23(1H，m)，7.27(4H，app?d)，11.27(1H，broad?s)。
MS(ESI，m/z)：437(M+Na，100％)
＜embodiment 8〉intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-phenyl-methane amide
Embodiment 3 gained intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-(15.0g 37.5mmol) is suspended among the 10% methanol solution 440mL of triethylamine phenyl-methane amide, stirs molten clear down in 30 ℃, decompression is spin-dried for after 3-5 hour, the residue grinding that adds diethyl ether, suction filtration, ether (40mL) washing, vacuum-drying obtains white powder solid 12.06g, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.44(9H，s)，4.32-4.34(2H，m)，4.38-4.42(2H，m)，7.46(3H，m)，7.94(2H，d)。
MS(ESI，m/z)：351(M+Na，100％)
＜embodiment 9〉intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-methane amide
According to embodiment 8 described methods; with embodiment 4 gained intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide stirs deprotection in 10% methanol solution of triethylamine and gets title compound; the pulverulent solids that is white in color, yield 96%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.44(9H，s)，4.34-4.37(2H，m)，4.00-4.35(2H，m)，7.27(2H，d)，7.88(2H，d)。
MS(ESI，m/z)：365(M+Na，100％)
＜embodiment 10〉intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 8 described methods; with embodiment 5 gained intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide stirs deprotection in 10% methanol solution of triethylamine and gets title compound; the pulverulent solids that is white in color, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.44(9H，s)，4.28-4.45(4H，m)，7.30(2H，t)，8.06(2H，m)，10.90(1H，broad?s)，12.34(1H，broad?s)。
MS(ESI，m/z)：369(M+Na，100％)
＜embodiment 11〉intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 8 described methods; with embodiment 6 gained intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide stirs deprotection in 10% methanol solution of triethylamine and gets title compound; the pulverulent solids that is white in color, yield 99%.
1H?NMR(400MHz，DMSO- )δppm：1.44(9H，s)，3.80(3H，s)，4.38-4.41(4H，m)，7.00(2H，d)，7.93(2H，d)，10.69(1H，broad?s)，12.17(1H，broad?s)。
MS(ESI，m/z)：381(M+Na，100％)
＜embodiment 12〉intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 8 described methods; with embodiment 7 gained intermediate N { 5-tertbutyloxycarbonyl-1-ethoxycarbonyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl }-1-benzyl-methane amide stirs deprotection in 10% methanol solution of triethylamine and gets title compound; the pulverulent solids that is white in color, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.41(9H，s)，3.58(3H，s)，4.05-4.09(2H，m)，4.29-4.36(2H，m)，7.23-7.32(5H，m)，10.63(1H，broad?s)，12.22(1H，broad?s)。
MS(ESI，m/z)：365(M+Na，100％)
＜embodiment 13〉intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
Commercially available propionyl chloride (0.72mL, 8.25mmol) in 10mL tetrahydrofuran (THF) (reflux to lay equal stress on steam obtain through hydrolith), under 0-5 ℃, slowly drop to embodiment 8 intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl-phenyl-methane amide (3.0g, 7.5mmol) with the 80mL tetrahydrofuran (THF) of DIEA (7.4mL) (reflux to lay equal stress on steam obtain through hydrolith) solution in, ice bath 5 hours, allow to rise to room temperature, stirring is spent the night.Reaction solution is spin-dried for, and resistates dissolves with ethyl acetate (50mL), saturated NaHCO 3Washing (10mL * 2), the organic layer anhydrous sodium sulfate drying, suction filtration, filtrate decompression is removed and is desolvated, and resistates is through silicagel column separation and purification (petrol ether/ethyl acetate=8: 1), and vacuum-drying gets white powder solid 1.85g, yield 64.2%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.45(9H，s)，1.46(3H，t)，3.00(2H，q)，4.73-4.49(2H，m)，4.59-4.62(2H，m)，7.49(2H，t)，7.59(1H，t)，7.94(2H，d)，11.38(1H，broad?s)。
MS(ESI，m/z)：407(M+Na，100％)
＜embodiment 14〉intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound, the pulverulent solids that is white in color, yield 87% with commercially available Benzoyl chloride and embodiment 8 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-phenyl-methane amide.
1H?NMR(400MHz，DMSO-d 6)δppm：1.46(9H，s)，4.53-4.55(2H，m)，4.69-4.74(2H，m)，7.50(2H，t)，7.60(3H，m)，7.68(1H，t)，8.01-8.03(2H，m)，8.10-8.11(2H，m)，11.35(1H，broad?s)。
MS(ESI，m/z)：455(M+Na，100％)
＜embodiment 15〉intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available isobutyryl chloride and embodiment 8 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-phenyl-methane amide, be pale brown toner powder solid, yield 65%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.16-1.22(6H，m)，1.45(9H，s)，3.65(1H，m)，4.47-4.50(2H，m)，4.59-4.63(2H，m)，7.50(2H，t)，7.60(1H，t)，8.02-8.04(2H，m)，11.38(1H，broad?s)。
MS(ESI，m/z)：421(M+Na，100％)
＜embodiment 16〉intermediate N { 5-tertbutyloxycarbonyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available ring third formyl chloride and embodiment 8 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-phenyl-methane amide, the pulverulent solids that is white in color, yield 80%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.09(2H，m)，1.16(2H，m)，1.44(9H，s)，2.89(1H，m)，4.49-4.52(2H，m)，4.56-4.60(2H，m)，7.50(2H，t)，7.60(1H，m)，8.02-8.04(2H，m)，11.40(1H，broad?s)。
MS(ESI，m/z)：419(M+Na，100％)
＜embodiment 17〉intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available propionyl chloride and embodiment 9 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide, the pulverulent solids that is white in color, yield 72%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，t)，1.45(9H，s)，2.36(3H，s)，2.99(2H，q)，4.46-4.49(2H，m)，4.58-4.61(2H，m)，7.28-7.30(2H，m)，7.92-7.95(2H，m)，11.28(1H，broad?s)。
MS(ESI，m/z)：421(M+Na，100％)
＜embodiment 18〉intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available Benzoyl chloride and embodiment 9 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide, the pulverulent solids that is white in color, yield 75%.
1H?NMR(400MHz，DMSO-
Figure BSA00000444100100131
δppm：1.45(9H，s)，2.49(3H，s)，4.48-4.50(2H，m)，4.59-4.63(2H，m)，7.30-7.67(4H，m)，7.81-7.94(5H，m)，11.15(1H，broad?s)。
MS(ESI，m/z)：469(M+Na，100％)
＜embodiment 19〉intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with homemade isobutyryl chloride and embodiment 9 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide, be pale brown toner powder solid, yield 42%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.20-1.22(6H，m)，1.44(9H，s)，2.36(3H，s)，3.65(1H，m)，4.46-4.49(2H，m)，4.58-4.62(2H，m)，7.31(2H，m)，7.92-7.95(2H，m)，11.28(1H，broad?s)。
MS(ESI，m/z)：435(M+Na，100％)
＜embodiment 20〉intermediate N { 5-tertbutyloxycarbonyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with propionyl chloride and embodiment 9 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide, be off-white powder shape solid, yield 70%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.10(2H，m)，1.17(2H，m)，1.44(9H，s)，2.37(3H，s)，4.48-4.51(2H，m)，4.56-4.60(2H，m)，7.29(2H，t)，7.93-7.96(2H，m)，11.31(1H，broad?s)。
MS(ESI，m/z)：433(M+Na，100％)
＜embodiment 21〉intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available propionyl chloride and embodiment 10 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide, the pulverulent solids that is white in color, yield 43%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，t)，1.44(9H，s)，3.00(2H，q)，4.46-4.49(2H，m)，4.59-4.62(2H，m)，7.33(2H，t)，8.08-8.13(2H，m)，11.47(1H，broads)。
MS(ESI，m/z)：425(M+Na，100％)
＜embodiment 22〉intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available Benzoyl chloride and embodiment 10 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide, the pulverulent solids that is white in color, yield 55%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.46(9H，s)，4.52-4.54(2H，m)，4.69-4.74(2H，m)，7.33(2H，t)，7.56(2H，t)，7.66(2H，t)，8.09-8.12(4H，m)，11.38(1H，broad?s)。
MS(ESI，m/z)：473(M+Na，100％)
＜embodiment 23〉intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available isobutyryl chloride and embodiment 10 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide, be pale brown toner powder solid, yield 28%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.20-1.22(6H，m)，1.44(9H，s)，3.64(1H，m)，4.47-4.49(2H，m)，4.59-4.63(2H，m)，7.34(2H，m)，8.09-8.13(2H，m)，11.42(1H，broad?s)。
MS(ESI，m/z)：439(M+Na，100％)
＜embodiment 24〉intermediate N { 5-tertbutyloxycarbonyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available propionyl chloride and embodiment 10 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide, be pale brown toner powder solid, yield 56%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.12(2H，m)，1.23(2H，m)，1.44(9H，s)，4.48-4.51(2H，m)，4.56-4.61(2H，m)，7.34(2H，t)，8.09-8.14(2H，m)，11.44(1H，broads)。
MS(ESI，m/z)：437(M+Na，100％)
＜embodiment 25〉intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
Commercially available propionyl chloride (0.673mL, 8.25mmol) in 10mL tetrahydrofuran (THF) (reflux to lay equal stress on steam obtain through hydrolith), under 0-5 ℃, slowly drop to embodiment 11 intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl-4-methoxyl group-phenyl-methane amide (2.5g, 6.98mmol) with the 80mL tetrahydrofuran (THF) of DIEA (7.0mL) (reflux to lay equal stress on steam obtain through hydrolith) solution in, ice bath 5 hours, allow to rise to room temperature, stirring is spent the night.Reaction solution is spin-dried for, and resistates dissolves with ethyl acetate (80mL), saturated NaHCO 3Washing (40mL * 2), organic layer emulsification and separate out with a large amount of flosss during washing is left standstill, suction filtration, vacuum-drying gets white powder solid 1.78g, yield 62%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，t)，1.45(9H，s)，3.00(2H，q)，3.82(3H，s)，4.46-4.48(2H，m)，4.59-4.62(2H，m)，7.01-7.03(2H，m)，8.02-8.05(2H，m)，11.21(1H，broad?s)。
MS(ESI，m/z)：437(M+Na，100％)
＜embodiment 26〉intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available Benzoyl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide, be pale yellow powder shape solid, yield 35%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.46(9H，s)，3.82(1H，m)，4.51-4.53(2H，m)，4.68-4.72(2H，m)，7.00-7.03(2H，m)，7.56(2H，t)，7.68(2H，t)，8.01-8.04(2H，m)，8.09-8.11(2H，m)，11.17(1H，broad?s)。
MS(ESI，m/z)：463(M+Na，100％)
＜embodiment 27〉intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available isobutyryl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide, be brown ceramic powder shape solid, yield 38%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.16-1.22(6H，m)，1.44(9H，s)，3.65(1H，m)，3.83(3H，s)，4.46-4.49(2H，m)，4.58-4.62(2H，m)，7.01(2H，m)，8.02-8.05(2H，m)，11.21(1H，broad?s)。
MS(ESI，m/z)：451(M+Na，100％)
＜embodiment 28〉intermediate N { 5-tertbutyloxycarbonyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available propionyl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide, the pulverulent solids that is white in color, yield 76%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.10(2H，m)，1.17(2H，m)，1.44(9H，s)，2.89(1H，m)，3.83(3H，s)，4.47-4.50(2H，m)，4.56-4.60(2H，m)，7.02-7.04(2H，m)，8.03-8.05(2H，m)，11.24(1H，broad?s)。
MS(ESI，m/z)：449(M+Na，100％)
＜embodiment 29〉intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 13 described methods, prepare title compound, white solid, yield 85% with commercially available propionyl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide.
1H?NMR(400MHz，DMSO-d 6)δppm：1.10(3H，m)，1.42(9H，s)，2.36(3H，s)，3.00(2H，m)，3.64(2H，s)，4.33-4.37.(2H，m)，4.52-4.56(2H，m)，7.21-7.33(5H，m)，11.17(1H，broad?s)。
MS(ESI，m/z)：421(M+Na，100％)
＜embodiment 30〉intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available Benzoyl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide, the white powder solid, yield 55%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.44(9H，s)，3.64(2H，s)，4.39-4.43(2H，m)，4.63-4.68(2H，m)，7.29-7.31(5H，m)，7.53-7.57(2H，m)，7.65-7.67(1H，m)，8.03-8.05(2H，m)，11.17(1H，broad?s)。
MS(ESI，m/z)：469(M+Na，100％)
＜embodiment 31〉intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available isobutyryl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide, pale yellow powder shape solid, yield 33%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.17-1.23(6H，m)，1.42(9H，s)，3.56(1H，m)，3.65(2H，s)，4.33-4.38(2H，m)，4.52-4.57(2H，m)，7.23-7.31(5H，m)，11.18(1H，broad?s)。
MS(ESI，m/z)：435(M+Na，100％)
＜embodiment 32〉intermediate N { 5-tertbutyloxycarbonyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 13 described methods, prepare title compound with commercially available propionyl chloride and embodiment 11 gained intermediate N { 5-tertbutyloxycarbonyl-4,6-dihydro-1H-pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide, the pulverulent solids that is white in color, yield 66%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.06(2H，m)，1.14(2H，m)，1.41(9H，s)，2.80(1H，m)，3.65(2H，s)，4.34-4.38(2H，m)，4.51-4.54(2H，m)，7.24-7.31(5H，m)，11.18(1H，broad?s)。
MS(ESI，m/z)：433(M+Na，100％)
＜embodiment 33〉intermediate N { 1-propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride
With embodiment 13 intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl }-phenyl-methane amide (1.85g; 4.8mmol) be dissolved in the 40mL exsiccant methylene dichloride, logical hydrogen chloride gas 3～5 hours has a large amount of white precipitates to generate; suction filtration; methylene dichloride (20mL) washing, filter cake vacuum-drying gets 1.48g, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，t)，3.02(2H，q)，4.47-4.50(2H，m)，4.59-4.63(2H，m)，7.48-7.61(3H，m)，8.01-8.03(2H，m)，11.39(1H，s)。
MS(ESI，m/z)：285(M ++1，100％)
＜embodiment 34〉intermediate N { 1-benzoyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 14 gained intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：4.49(2H，m)，4.67(2H，m)，7.48-7.52(2H，m)，7.56-7.62(3H，m)，7.68-7.72(1H，m)，7.97-8.02(2H，m)，8.10-8.13(2H，m)，10.15(1H，broad?s)，11.51(1H，s)。
MS(ESI，m/z)：333(M ++1，100％)
＜embodiment 35〉intermediate N { 1-isobutyryl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 15 gained intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.16-1.18(6H，m)，3.57(1H，m)，4.31-4.34(2H，m)，4.50-4.54(2H，m)，7.50-7.63(3H，m)，8.04-8.06(2H，m)，11.39(1H，s)。
MS(ESI，m/z)：299(M ++1，100％)
＜embodiment 36〉intermediate N { 1-cyclopropane carbonyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 16 gained intermediate intermediate N { 5-tertbutyloxycarbonyls-1-cyclopropane carbonyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.12(2H，m)，1.24(2H，m)，2.87(1H，m)，4.46(2H，m)，4.55(2H，m)，7.52-7.64(3H，m)，8.03-8.05(2H，m)，9.94(1H，broad?s)，11.59(1H，s)。
MS(ESI，m/z)：297(M ++1，100％)
＜embodiment 37〉intermediate N { 1-propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 17 gained intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methyl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.15(3H，t)，2.37(3H，s)，3.03(2H，q)，4.44(2H，m)，4.57(2H，m)，7.30-7.32(2H，m)，7.89-7.95(2H，m)，10.13(1H，broads)，11.49(1H，s)。
MS(ESI，m/z)：299(M ++1，100％)
＜embodiment 38〉intermediate N { 1-benzoyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 18 gained intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methyl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：2.37(3H，s)，4.39(2H，m)，4.67(2H，m)，7.30-7.32(2H，m)，7.57-7.60(2H，m)，7.69-7.73(1H，m)，7.89-7.95(2H，m)，8.12-8.13(2H，m)，10.16(1H，broad?s)，11.43(1H，s)。
MS(ESI，m/z)：347(M ++1，100％)
＜embodiment 39〉intermediate N { 1-isobutyryl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 19 gained intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methyl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.20-1.22(6H，m)，3.60(1H，m)，2.39(3H，s)，4.49(2H，m)，4.67(2H，m)，7.35-7.37(2H，m)，8.00-8.06(2H，m)，10.41(1H，broad?s)，11.69(1H，s)。
MS(ESI，m/z)：313(M ++1，100％)
＜embodiment 40〉intermediate N { 1-encircles propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; { 5-tertbutyloxycarbonyl-1-encircles propionyl-4 with embodiment 20 gained intermediate N; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methyl-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.12(2H，m)，1.22(2H，m)，2.37(3H，s)，2.87(1H，m)，4.45(2H，m)，4.55(2H，m)，7.31-7.33(3H，m)，7.93-7.95(2H，m)，10.00(1H，broad?s)，11.50(1H，s)。
MS(ESI，m/z)：311(M ++1，100％)
＜embodiment 41〉intermediate N { 1-propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 21 gained intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-fluoro-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，t)，3.00(2H，q)，4.45(2H，m)，4.46-4.49(2H，m)，4.59-4.62(2H，m)，7.31-7.35(3H，m)，8.08-8.13(2H，m)，11.42(1H，s)。
MS(ESI，m/z)：303(M ++1，100％)
＜embodiment 42〉intermediate N { 1-benzoyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 22 gained intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-fluoro-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：4.38-4.40(2H，m)，4.49-4.68(2H，m)，7.33-7.37(3H，m)，7.57-7.60(3H，m)，7.68-7.73(1H，m)，8.06-8.13(4H，m)，10.25(1H，broad?s)，11.55(1H，s)。
MS(ESI，m/z)：351(M ++1，100％)
＜embodiment 43〉intermediate N { 1-isobutyryl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 23 gained intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-fluoro-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.21-1.23(6H，m)，3.63(1H，m)，4.46(2H，m)，4.55(2H，m)，7.33-7.37(3H，m)，8.00-8.03(2H，m)，10.02(1H，broad?s)，11.32(1H，s)。
MS(ESI，m/z)：317(M ++1，100％)
＜embodiment 44〉intermediate N { 1-encircles propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; { 5-tertbutyloxycarbonyl-1-encircles propionyl-4 with embodiment 24 gained intermediate N; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-fluoro-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.12(2H，m)，1.22(2H，m)，2.85(1H，m)，4.45(2H，m)，4.55(2H，m)，7.34-7.38(3H，m)，8.10-8.13(2H，m)，10.10(1H，broads)，11.62(1H，s)。
MS(ESI，m/z)：315(M ++1，100％)
＜embodiment 45〉intermediate N { 1-propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 25 gained intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methoxyl group-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.14(3H，m)，3.01(1H，m)，3.82(3H，s)，4.43-4.49(2H，m)，4.56-4.62(2H，m)，7.01-7.05(2H，m)，8.02-8.04(2H，m)，9.99(1H，broad?s)，11.21(1H，s)。
MS(ESI，m/z)：315(M ++1，100％)
＜embodiment 46〉intermediate N { 1-benzoyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 26 gained intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methoxyl group-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.82(3H，s)，4.49(2H，m)，4.67(2H，m)，7.02-7.05(2H，m)，7.57-7.60(2H，m)，7.69-7.73(1H，m)，7.97-8.04(2H，m)，8.11-8.13(2H，m)，10.16(1H，broad?s)，11.34(1H，s)。
MS(ESI，m/z)：363(M ++1，100％)
＜embodiment 47〉intermediate N { 1-isobutyryl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 27 gained intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methoxyl group-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.21-1.23(6H，m)，3.63(1H，m)，3.82(3H，s)，4.28-4.31(2H，m)，4.37-4.43(2H，m)，7.03-7.05(2H，m)，7.98-8.05(2H，m)，9.92(1H，broad?s)，11.01(1H，s)。
MS(ESI，m/z)：329(M ++1，100％)
＜embodiment 48〉intermediate N { 1-encircles propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride
According to embodiment 33 described methods; { 5-tertbutyloxycarbonyl-1-encircles propionyl-4 with embodiment 28 gained intermediate N; 6-pyrrolin [3; 4-c] pyrazole-3-yl-4-methoxyl group-phenyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.11-1.23(4H，m)，2.87(1H，m)，3.83(3H，s)，4.44(2H，m)，4.54(2H，m)，7.03-7.05(2H，m)，8.03-8.05(2H，m)，9.78(1H，broads)，11.41(1H，s)。
MS(ESI，m/z)：327(M ++1，100％)
＜embodiment 49〉intermediate N { 1-propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 29 gained intermediate N { 5-tertbutyloxycarbonyl-1-propionyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-1-benzyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.13(3H，t)，2.96(2H，q)，3.66(2H，s)，4.30(2H，m)，4.50(2H，m)，7.24-7.31(5H，m)，9.93(1H，broad?s)，11.36(1H，s)。
MS(ESI，m/z)：299(M ++1，100％)
＜embodiment 50〉intermediate N { 1-benzoyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 30 gained intermediate N { 5-tertbutyloxycarbonyl-1-benzoyl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-1-benzyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.66(1H，m)，4.25-4.36(2H，m)，4.62(2H，m)，7.23-7.33(5H，m)，7.56-7.60(5H，m)，7.68-7.72(5H，m)，8.05-8.07(5H，m)，10.09(1H，broad?s)，11.37(1H，s)。
MS(ESI，m/z)：347(M ++1，100％)
＜embodiment 51〉intermediate N { 1-isobutyryl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride
According to embodiment 33 described methods; with embodiment 31 gained intermediate N { 5-tertbutyloxycarbonyl-1-isobutyryl-4; 6-pyrrolin [3; 4-c] pyrazole-3-yl-1-benzyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.19-1.20(6H，m)，3.54(1H，m)，4.25-4.30(2H，m)，4.50(2H，m)，7.22-7.33(5H，m)，9.88(1H，broad?s)，11.37(1H，s)。
MS(ESI，m/z)：313(M ++1，100％)
＜embodiment 52〉intermediate N { 1-encircles propionyl-4,5,6-three hydrogen pyrroles [3,4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride
According to embodiment 33 described methods; { 5-tertbutyloxycarbonyl-1-encircles propionyl-4 with embodiment 32 gained intermediate N; 6-pyrrolin [3; 4-c] pyrazole-3-yl-1-benzyl-methane amide in methylene dichloride, feed hydrochloric acid qi exhaustion Boc protect title compound; the white powder solid, yield 98%.
1H?NMR(400MHz，DMSO-d 6)δppm：1.08-1.22(4H，m)，2.78(1H，m)，3.67(2H，s)，4.31(2H，m)，4.48(2H，m)，7.23-7.33(2H，m)，9.92(1H，broad?s)，11.38(1H，s)。
MS(ESI，m/z)：311(M ++1，100％)
＜embodiment 53〉N-{5-benzoyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
Scheme 1
(a) 1,4,5, the preparation of 6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride
Embodiment 1 compound 3-amino-4,6-dihydro-1H-pyrroles [3,4-c] pyrazoles-5-carboxylic acid tertiary butyl ester 4.0g, 17.8mmol be dissolved in the 60mL exsiccant methylene dichloride, logical hydrogen chloride gas 3～5 hours has a large amount of white precipitates to generate, suction filtration, methylene dichloride (20mL) washing, filter cake vacuum-drying gets 1,4,5,6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride 2.85g, yield 100%.
1H?NMR(400MHz，DMSO-d 6)δppm：4.12(2H，m)，4.29(2H，m)，10.36(1H，broad?s)。
MS(ESI，m/z)：125(M ++1，100％)。
(b) N-{5-benzoyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-preparation of phenyl-methane amide
(4.35mL 37.5mmol) is dissolved among the anhydrous THF of 20mL N with commercially available Benzoyl chloride 2Protection slowly drops to embodiment 53 (a) gained 1,4; 5, in 6-Pyrrolidine [3, the 4-c] pyrazoles-DIEA of 3-semicarbazide hydrochloride 2.0g (12.5mmol), 37.2mL (225mmol) and the mixture of the anhydrous THF of 100mL; stirred 6 hours under the room temperature, be spin-dried for, methylene dichloride (100mL) extraction; washing; anhydrous sodium sulfate drying, suction filtration, filtrate is spin-dried for; get white powder solid 4.8g, yield 88%.
1H?NMR(400MHz，DMSO-d 6)δppm：4.70-4.98(4H，m)，7.43-7.94(12H，m)，7.96-8.14(5H，m)，11.39(1H，s)。
MS(ESI，m/z)：459(M+Na，100％)
Scheme 2
With commercially available Benzoyl chloride (0.21g or 173 μ L; 1.5mmol) methylene dichloride (5mL) mixed solution be added dropwise to embodiment 34 gained intermediate N { 1-benzoyls-4; 5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride (0.5g; 1.36mmol) methylene dichloride (20mL) and DIEA (1.35mL; 8.16mmol) suspension in, stirred overnight at room temperature, saturated common salt washing; the anhydrous sodium sulfate drying organic phase; be spin-dried for, crude product gets title compound through silicagel column separation and purification (petrol ether/ethyl acetate=6: 1); white powder solid 0.46g, yield 78%.
1H?NMR(400MHz，DMSO-d 6)δppm：4.70-4.98(4H，m)，7.43-7.94(12H，m)，7.96-8.13(5H，m)，11.39(1H，s)。
MS(ESI，m/z)：459(M+Na，100％)
＜embodiment 54〉N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
With n-butyl isocyanate (112 μ L; 1.0mmol) methylene dichloride (5mL) mixed solution be added dropwise to embodiment 33 gained intermediate N { 1-propionyls-4; 5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride (0.20g; 0.625mmol) methylene dichloride (20mL) and DIEA (0.83mL; 5.0mmol) suspension in, stirred overnight at room temperature, saturated common salt washing; the anhydrous sodium sulfate drying organic phase; be spin-dried for, crude product gets title compound through silicagel column separation and purification (petrol ether/ethyl acetate=6: 1); white powder solid 0.20g, yield 80%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.88(3H，m)，1.15(3H，m)，1.45(6H，m)，3.02(2H，q)，4.47-4.50(4H，m)，4.60-4.63(6H，m)，7.48-8.61(3H，m)，8.01-8.04(2H，m)，11.39(1H，s)。
MS(ESI，m/z)：407(M+Na，100％)
＜embodiment 55〉N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 34 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 70%.
1H?NMR(400MHz，DMSO-
Figure BSA00000444100100251
δppm：0.88(3H，t)，1.30(2H，m)，1.43(2H，m)，3.05(2H，m)，4.36-4.72(4H，m),7.48-7.69(6H，m)7.87-8.11(4H，m)11.33(1H，s)。
MS(ESI，m/z)：454(M+Na，100％)
＜embodiment 56〉N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 36 gained intermediate N { 1-cyclopropane carbonyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 90%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.86(3H，t)，1.06-1.08(4H，m)，1.17(2H，m)，1.34(2H，m)，2.81(1H，m)，2.99(2H，m)，4.37-4.38(2H，m)，4.53-4.54(2H，m)，7.24-7.32(5H，m)，11.22(1H，s)。
MS(ESI，m/z)：396(M ++1，100％)
＜embodiment 57〉N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 37 gained intermediate N { 1-propionyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 76%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.86(3H，m)，1.13(2H，m)，1.17(3H，m)，1.19(2H，m)，3.00(1H，m)，3.65(2H，m)，4.40-4.42(4H，m)，7.11-7.13(2H，m)，8.12-8.15(2H，m)，11.32(1H，s)。
MS(ESI，m/z)：420(M+Na，100％)
＜embodiment 58〉N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 38 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 68%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.87(3H，t)，1.22-1.27(2H，m)，1.31-1.37(2H，m)，2.35(3H，s)，3.64(3H，m)，4.41-4.42(2H，m)，4.52(2H，m)，7.08-7.11(2H，m)，8.08-8.12(2H，m)，11.19(1H，s)。
MS(ESI，m/z)：468(M+Na，100％)
＜embodiment 59〉N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 40 gained intermediate N { 1-cyclopropane carbonyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 68%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.86(3H，t)，1.07-1.17(4H，m)，1.23-1.29(2H，m)，1.36-1.42(2H，m)，2.36(3H，s)，2.90(1H，m)，3.82(3H，s)，4.47-4.48(2H，m)，4.57(2H，m)，7.01-7.03(2H，m)，8.01-8.04(2H，m)，11.13(1H，s)。
MS(ESI，m/z)：422(M+Na，100％)
＜embodiment 60〉N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 41 gained intermediate N { 1-propionyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 67%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.85(3H，m)，1.12(2H，m)，1.16(3H，m)，1.18(2H，m)，3.04(3H，m)，3.82(2H，s)，4.40-4.42(4H，m)，7.11-7.13(2H，m)，8.12-8.15(2H，m)，11.32(1H，s)。
MS(ESI，m/z)：422(M+Na，100％)
＜embodiment 61〉N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 42 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 78%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.86(3H，t)，1.26(2H，m)，1.40(2H，m)，3.00(2H，m)，4.44(2H，m)，4.56(2H，m)，7.00-7.02(3H，m)，7.52-7.56(2H，m)，7.64-7.65(1H，m)，8.01-8.09(4H，m)，11.14(1H，s)。
MS(ESI，m/z)：472(M ++1，100％)
＜embodiment 62〉N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 54 described methods, { 1-encircles propionyl-4,5 to use n-butyl isocyanate and embodiment 44 gained intermediate N; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 60%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.86(3H，t)，1.08-1.18(4H，m)，1.19-1.32(2H，m)，1.37-1.44(2H，m)，2.90(1H，m)，3.03(2H，m)，4.49-4.50(2H，m)，4.59(2H，m)，7.33-7.37(2H，m)，8.10-8.14(2H，m)，11.43(1H，s)。
MS(ESI，m/z)：436(M+Na，100％)
＜embodiment 63〉N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 43 gained intermediate N { 1-isobutyryl-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 70%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.88(3H，t)，1.21-1.22(6H，m)，1.25-1.35(2H，m)，1.46-1.54(2H，m)，3.05(1H，m)，4.37-4.48(2H，m)，4.58-4.61(2H，m)，7.32-7.47(2H，m)，7.93-8.13(2H，m)，11.40(1H，s)。
MS(ESI，m/z)：438(M+Na，100％)
＜embodiment 64〉N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 45 gained intermediate N { 1-propionyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 73%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.84(3H，m)，1.14(3H，m)，1.29(4H，m)，2.93-3.04(3H，m)，3.82(2H，s)，4.46-4.48(4H，m)，7.01-7.03(2H，m)，8.02-8.05(2H，m)，11.22(1H，s)。
MS(ESI，m/z)：437(M+Na，100％)
＜embodiment 65〉N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 46 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 66%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.88(3H，t)，1.28(2H，m)，1.43(2H，m)，3.05(2H，m)，3.82(3H，s)，4.52(2H，m)，4.71(2H，m)，7.01-7.03(3H，m)，7.54-7.58(2H，m)，7.65-7.66(1H，m)，8.02-8.10(4H，m)，11.16(1H，s)。
MS(ESI，m/z)：484(M+Na，100％)
＜embodiment 66〉N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 48 gained intermediate N { 1-cyclopropane carbonyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 68%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.87(3H，t)，1.08-1.18(4H，m)，1.24-1.30(2H，m)，1.37-1.43(2H，m)，2.91(1H，m)，3.03(2H，m)，3.83(3H，s)，4.48-4.49(2H，m)，4.59(2H，m)，7.02-7.04(2H，m)，8.03-8.06(2H，m)，11.23(1H，s)。
MS(ESI，m/z)：448(M+Na，100％)
＜embodiment 67〉N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 47 gained intermediate N { 1-isobutyryl-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 65%.
1H?NMR(400MHz，DMSO- )δppm：0.86(3H，m)，1.20-1.21(6H，m)，1.25(2H，m)，1.39(2H，m)，3.05(1H，m)，3.83(3H，s)，4.36-4.47(2H，m)，4.58-4.60(2H，m)，7.02-7.15(3H，m)，8.03-8.05(4H，m)，11.20(1H，s)。
MS(ESI，m/z)：450(M+Na，100％)
＜embodiment 68〉N-{5-normal-butyl aminocarboxyl-1-propionyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 49 gained intermediate N { 1-propionyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 70%.
1H?NMR(400MHz，DMSO-d 6).δppm：0.85(3H，m)，1.24(3H，m)，1.34(2H，m)，1.38(2H，m)，2.99(2H，m)，3.65(2H，s)，4.36(2H，m)，4.54(2H，m)，7.21-7.33(5H，m)，11.16(1H，s)。
MS(ESI，m/z)：398(M+Na，100％)
＜embodiment 69〉N-{5-normal-butyl aminocarboxyl-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 50 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 78%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.87(3H，t)，1.23(2H，m)，1.33(2H，m)，3.00(2H，m)，3.65(2H，s)，4.41-4.44(2H，m)，4.66(2H，m)，7.30-7.35(6H，m)，7.47-7.53(2H，m)，7.65-7.69(1H，m)，8.03-8.04(2H，m)，11.16(1H，s)。
MS(ESI，m/z)：468(M+Na，100％)
＜embodiment 70〉N-{5-normal-butyl aminocarboxyl-1-cyclopropane carbonyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 52 gained intermediate N { 1-cyclopropane carbonyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 45%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.85(3H，m)，1.06-1.08(4H，m)，1.22-1.27(2H，m)，1.29-1.40(2H，m)，2.99(2H，m)，3.64(2H，s)，4.29-4.52(4H，m)，7.21-7.32(5H，m)，11.17(1H，s)。
MS(ESI，m/z)：422(M+Na，100％)
＜embodiment 71〉N-{5-normal-butyl aminocarboxyl-1-isobutyryl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 54 described methods, with n-butyl isocyanate and embodiment 51 gained intermediate N { 1-isobutyryl-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 60%.
1H?NMR(400MHz，DMSO-d 6)δppm：0.85(3H，m)，1.18-1.20(6H，m)，1.23-1.28(2H，m)，1.30-1.41(2H，m)，3.01(1H，m)，3.57(1H，m)，3.66(2H，s)，4.30-4.54(4H，m)，7.23-7.33(5H，m)，11.16(1H，s)。
MS(ESI，m/z)：434(M+Na，100％)
＜embodiment 72〉N-{5-(4-methyl-benzoyl)-1-(4-methyl-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to the described method of embodiment 53 schemes 1 (b), with commercially available Butyltriphenylphosphonium chloride and embodiment 53 scheme 1 (a) gained 1,4,5,6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 80%.
1H?NMR(400MHz，DMSO-d 6)δppm：2.33(9H，m)，4.71-4.99(4H，m)，7.03-7.54(8H，m)，7.66-8.03(4H，m)，11.45(1H，s)。
MS(ESI，m/z)：501(M+Na，100％)
＜embodiment 73〉N-{5-(4-methoxyl group-benzoyl)-1-(4-methoxyl group-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to the described method of embodiment 53 schemes 1 (b), with commercially available anisoyl chloride and embodiment 53 scheme 1 (a) gained 1,4,5,6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 76%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.80(9H，m)，4.49-4.50(2H，m)，4.60(2H，m)，7.12-7.34(8H，m)，7.53-7.76(4H，m)，11.13(1H，s)
MS(ESI，m/z)：549(M+Na，100％)
＜embodiment 74〉N-{5-(4-fluoro-benzoyl)-1-(4-fluoro-benzoyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to the described method of embodiment 53 schemes 1 (b), to fluorobenzoyl chloride and embodiment 53 scheme 1 (a) gained 1,4,5,6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 79% with commercially available.
1H?NMR(400MHz，DMSO-d 6)δppm：4.36-4.46(2H，m)，4.56-4.59(2H，m)，7.22-7.50(8H，m)，7.83-8.03(4H，m)，11.41(1H，s)。
MS(ESI，m/z)：513(M+Na，100％)
＜embodiment 75〉N-{5-(1-benzyl)-1-(1-benzyl)-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to the described method of embodiment 53 schemes 1 (b), with commercially available phenyllacetyl chloride and embodiment 53 scheme 1 (a) gained 1,4,5,6-Pyrrolidine [3,4-c] pyrazoles-3-semicarbazide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 80%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.60(6H，m)，4.40-4.43(2H，m)，4.64(2H，m)，7.30-7.55(8H，m)，7.67-7.89(4H，m)，11.11(1H，s)。
MS(ESI，m/z)：501(M+Na，100％)
＜embodiment 76〉N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methyl-phenyl-methane amide
According to embodiment 54 described methods, with commercially available Benzoyl chloride and embodiment 38 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methyl-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 78%.
1H?NMR(400MHz，DMSO-d 6)δppm：2.30(3H，s)，4.71-4.99(4H，m)，7.40-7.90(6H，m)，7.91-8.12(3H，m)，11.43(1H，s)。
MS(ESI，m/z)：473(M+Na，100％)
＜embodiment 77〉N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-methane amide
According to embodiment 54 described methods, with commercially available Benzoyl chloride and embodiment 46 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-methoxyl group-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 81%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.81(3H，s)，4.45-4.76(4H，m)，7.21-7.54(6H，m)，7.65-8.00(3H，m)，11.23(1H，s)。
MS(ESI，m/z)：489(M+Na，100％)
＜embodiment 78〉N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-4-fluoro-phenyl-methane amide
According to embodiment 54 described methods, with commercially available Benzoyl chloride and embodiment 42 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-4-fluoro-phenyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 80%.
1H?NMR(400MHz，DMSO-d 6).δppm：3.81(3H，s)，4.67-4.90(4H，m)，7.33-7.65(6H，m)，7.76-8.03(3H，m)，11.12(1H，s)。
MS(ESI，m/z)：477(M+Na，100％)
＜embodiment 79〉N-{5-(1-benzoyl)-1-benzoyl-4,6-pyrrolin [3,4-c] pyrazole-3-yl }-1-benzyl-methane amide
According to embodiment 54 described methods, with commercially available Benzoyl chloride and embodiment 50 gained intermediate N { 1-benzoyls-4,5; 6-three hydrogen pyrroles [3; 4-c] pyrazole-3-yl }-1-benzyl-carboxamide hydrochloride prepares title compound, the pulverulent solids that is white in color, yield 85%.
1H?NMR(400MHz，DMSO-d 6)δppm：3.64(2H，s)，4.40-4.43(2H，m)，4.61(2H，m)，7.31-7.69(6H，m)，7.89-8.04(3H，m)，11.06(1H，s)。
MS(ESI，m/z)：473(M+Na，100％)
＜embodiment 80〉evaluation of The compounds of this invention antitumor efficacy
Compound of the present invention is carried out on the pharmacology experiment at human colon cancer cell HCT116, and (sulforhodam ine B SRB), as positive control drug, calculates the IC50 of growth of tumour cell with Rescovitine to adopt sulfo group rhodamine B method.According to growth of tumour cell speed, the human colon cancer cell HCT116 that will be in logarithmic phase is inoculated in 96 well culture plates with 200 μ L/ holes, each porocyte density is 4000/ hole, behind the adherent growth 24h, add embodiment compound 53 respectively), 54), 55), 56), 57), 58), 59), 60), 61), 62), 63), 64), 65), 66), 67), 68), 69), 70), 71), each concentration is established 3 multiple holes, and establishes the contrast of physiological saline solvent and the cell zeroing hole of respective concentration.Tumour cell is at 37 ℃, 5%CO 2Cultivate 48h under the condition.Take out culture plate, every hole adds trichoroacetic acid(TCA) (TCA) the 50 μ L fixed cells of 50% (m/v), places 1h for 4 ℃.Abandon stationary liquid, with distilled water wash 5 times, seasoning in the air, every hole adds SRB solution (0.4% sulfo group rhodamine B solution of 1% acetic acid preparation, the sulfo group rhodamine B be a kind of can be specifically under acidic conditions and intracellular protein bonded dyestuff) 100 μ L, place 10-30min under the room temperature, remove supernatant liquid, with 1% acetic acid washing 5 times, dry air, add the Tris buffered soln (Tutofusin tris buffered soln, pH 7.2～9.0) in 150 μ L/ holes at last, 5min vibrates on dull and stereotyped device.(Model3550 Bio-Rad) measures the OD value, and with the blank zeroing, used wavelength is 492nm at enzyme-linked immunosorbent assay instrument.
Table 1 representative compounds of the present invention is to the IC50 of CCL188 HCT116
Figure BSA00000444100100331
Figure BSA00000444100100341
Figure BSA00000444100100351
Figure BSA00000444100100361
Experimental result shows that all test compounds all have remarkable restraining effect to tumour cell in the table 1, and IC50 is near positive control drug on cell levels for most compounds, and especially embodiment 53 compounds obviously are better than positive control.

Claims (7)

1. the mixture of bicyclic amino group pyrazole compound and isomer thereof, steric isomer or steric isomer and pharmacy acceptable salt and precursor, its structure is shown in general formula (I):
Figure FSA00000444090000011
In the formula:
R 1Independently be selected from: C 1-C 10Alkyl, C 2-C 10Alkenyl, C 2-C 10Alkynyl group, C 3-C 8Cycloalkyl, C 4-C 12Cycloalkylalkyl, aryl, heteroaryl ,-COR 4,-COOR 4,-CONHR 4,-CONR 4R 5,-C (=NH) NHR 4,-SO 2R 4,-SO 2NHR 4,-SO 2NR 4R 5
R 2, R 3The independent hydrogen atom or optional of representing from R 4,-COR 4,-COOR 4,-CONHR 4,-CONR 4R 5,-C (=NH) NHR 4,-SO 2R 4,-SO 2NHR 4,-SO 2NR 4R 5
M and n represent 1,2 separately;
C 1-C 10Alkyl, C 2-C 10Alkenyl, C 2-C 10Alkynyl group, C 3-C 8Cycloalkyl, C 4-C 12Cycloalkylalkyl is optional independently to be selected from following substituting group replacement: C by 1-3 1-C 4Alkyl, nitro, halogen, cyano group, NR 4R 5, NR 4COR 5, NR 4CO 2R 5, COR 4, OR 4, CONR 4R 5, CO 2R 4
R 4And R 5Independently be selected from hydrogen atom or C 1-C 6Alkyl, C 3-C 6Cycloalkyl, C 4-C 6Alkenyl, C 4-C 6Alkynyl group, aryl, heteroaryl;
Aryl is a phenyl or naphthyl, and each is chosen wantonly by 1-5 and independently is selected from following substituting group replacement: C 1-C 6Alkyl, C 3-C 6Cycloalkyl, halogen, C 1-C 4Haloalkyl, cyano group, nitro ,-NR 6R 7,-CONR 6R 7,-NR 6COR 8,-OR 9,-COOR 9,-SR 9,-SO 2R 9,-SO 2NR 6R 7,-NR 6SO 2R 9
Heteroaryl is pyridyl, pyrimidyl, triazinyl, furyl, pyranyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indyl, pyrryl, oxazolyl, benzofuryl, benzothiazolyl, isoxazolyl, pyrazolyl, 2,3-dihydrobenzo thienyl or 2, the 3-dihydro benzo furyl, each is chosen wantonly by 1～5 and independently is selected from following substituting group replacement: C 1-C 6Alkyl, C 3-C 6Cycloalkyl, halogen, C 1-C 4Haloalkyl, cyano group, nitro ,-NR 6R 7,-CONR 6R 7,-NR 9COR 8,-OR 9,-COOR 9,-SR 9,-SO 2R 9,-SO 2NR 6R 7,-NR 6SO 2R 9
R 6, R 7, R 8, R 9Independent separately hydrogen atom or the C of representing 1-C 6Alkyl or phenyl.
2. the mixture of compound as claimed in claim 1 and isomer, steric isomer or steric isomer and pharmacy acceptable salt or precursor, it is characterized in that, the compound of general formula (I) exists with geometry and optical isomer, and all isomer are isolating, pure or partially purified steric isomer or its racemic mixture.
3. compound as claimed in claim 1 or 2 and isomer thereof, the mixture of steric isomer or steric isomer and pharmacy acceptable salt or precursor, it is characterized in that, the compound of general formula (I) is optionally with pharmaceutically-acceptable acid addition, metal-salt or selective alkylation ammonium salt exist, comprise inorganic and organic acid addition salt-hydrochloride, hydrobromate, vitriol, phosphoric acid salt, acetate, trifluoroacetate, succinate, fumarate, maleate, Citrate trianion, lactic acid salt, tartrate, mandelate, oxalate or similar pharmaceutically acceptable inorganic or organic acid addition salt, the perhaps an alkali metal salt that obtains of method for transformation routinely, alkaline earth salt, silver salt and barium salt.
4. prepare the described compound of claim 1 and the mixture of isomer, steric isomer or steric isomer and the method for pharmacy acceptable salt and precursor, it comprises the steps:
1) starting raw material II and acethydrazide be after reacting 2～24 hours under 0 ℃～100 ℃ in propyl carbinol, again with N methyl piperazine back flow reaction 8～24 hours, intermediate III;
2) there is acid acceptor to exist down, III and Vinyl chloroformate in polar aprotic solvent, were reacted 0.5～24 hour down at-10 ℃～30 ℃, obtain intermediate compound IV;
3) have acid acceptor to exist down, with IV in polar aprotic solvent ,-10 ℃ to reflux temperature, obtain intermediate V with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively;
4) with V in 10% triethylamine methanol solution, in-10 ℃ to down reaction 0.5～24 hour of reflux temperature, obtain intermediate VI;
5) have acid acceptor to exist down, with VI in polar aprotic solvent ,-10 ℃ to reflux temperature, obtain intermediate VII with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively;
6) with VII in anhydrous organic solvent, under acidic conditions, reacted 1～24 hour, obtain intermediate VIII;
7) have acid acceptor to exist down, with VIII in polar aprotic solvent ,-10 ℃ to reflux temperature, obtain formula (I) compound with acylting agent, alkylsulfonyl reagent, alkylating reagent reaction respectively.
5. claim 1, the application of 2 or 3 described compounds in the preparation antitumor drug.
6. pharmaceutical composition, it comprises claim 1,2 or 3 described compounds and pharmaceutically acceptable carrier.
7. the application of the described composition of claim 6 in the preparation antitumor drug.
CN2011100520021A 2011-03-04 2011-03-04 Bicyclic amino pyrazole derivatives with anti-tumor activity Pending CN102134243A (en)

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