CN101591285B - Propionamides compound and usage thereof - Google Patents
Propionamides compound and usage thereof Download PDFInfo
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- CN101591285B CN101591285B CN2008100380664A CN200810038066A CN101591285B CN 101591285 B CN101591285 B CN 101591285B CN 2008100380664 A CN2008100380664 A CN 2008100380664A CN 200810038066 A CN200810038066 A CN 200810038066A CN 101591285 B CN101591285 B CN 101591285B
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Abstract
The invention relates to a propionamides compound and a usage thereof. The inventor, by carrying out SPR experiment based on Biacore and making comprehensive use of CADD, modifies the structure of a propionamide derivative and obtains an N-substituted-2-substituted amino-3-(1H-indole-3-yl) propionamides compound (the structure thereof is indicated by formula I). The falcipain-2 activity inhibiting capability of the obtained compound is tested, and the result shows that the prepared N-substituted-2-substituted amino-3-(1H-indole-3-yl) propionamides compound has strong falcipain-2 activity inhibiting capability and can be used for preparing medicaments for treating malaria caused by plasmodium.
Description
Technical field
The present invention relates to a kind of Propionamides compound and uses thereof, specifically, relate to N-replacement-2-substituted-amino-3-(1H-indol-3-yl) Propionamides compound and uses thereof.
Background technology
Malaria is that one of more frequent parasitosis takes place on the earth, is the disease of being propagated, had potential fatal risk by anopheles.Along with the chemical sproof continuous increase of existing antimalarial agent, the sickness rate of malaria increases day by day, demands having the discovery of the antimalarial agent of novel therapeutic effect urgently.
The plasmodium of erythrocytic phase hydrolysis host's in its acidic food bubble oxyphorase is to obtain required energy and the amino acid of self life.Biological study shows, forgives a series of lytic enzyme in plasmodial food vacuole, as aspartate protease (plasmepsins), and L-Cysteine HCL Anhydrous (falcipains), and metalloprotease (falcilysins).These enzymes have become the chemotherapeutical potential target of malaria.
L-Cysteine HCL Anhydrous is M
τBe about 21000～30000 protein, o'clock have the highest hydrolytic activity in pH 4～6.5, its reactive site has cysteine residues.Plasmodial L-Cysteine HCL Anhydrous belongs to papoid family.Known plasmodium L-Cysteine HCL Anhydrous has four hypotypes, falcipain-1, falcipain-2A, falcipain-2B, falcipain-3.Falcipain1 is the plasmodium L-Cysteine HCL Anhydrous that first expression obtains, and its biological study shows that it is to plasmodial monogony stage did not influence, but the function of energy remarkable influence egg capsule.Falcipain-2A, falcipain-2B have 97% homology, and be only different 7 of aminoacid sequence.Monitoring by oligonucleotide probe finds that the expression level of falcipain-2B mRNA is lower than falcipain-2A.Yet falcipain-2A is very similar with peak value at the time-dependent manner of plasmodium nourishing body its expression in late period with falcipain-2B, and this shows that two kinds of different hypotypes have similar biological function.Falcipain-3 and falcipain-2 have 66.6% homology at catalytic domain, but the stage that they are expressed is different.Falcipain-2 reaches the climax at vegetative stage, and falcipain-3 is in that more the sophisticated plasmodium stage peaks.In these several hypotypes, maximum for the research of falcipain-2, so the exploitation of its inhibitor also is subjected to paying close attention to more widely.
In recent years, " area of computer aided medicinal design " (Computer-Aided Drug Design CADD) has become the important method and the instrument of modern medicines research and development.Its ultimate principle is: the utilization supercomputer by the method for molecular docking in reaching millions of micromolecular compound storehouses, seeks can with a certain specified disease target proteins bonded chemical structure, for further molecule, cell, animal and clinical study provide high-efficiency reliable small molecules lead compound.
In sum, as target, in conjunction with the biological activity test test of CADD and various levels, the micromolecular compound that designs and synthesizes has important practical significance to the exploitation antimalarial agent with L-Cysteine HCL Anhydrous falcipain-2.
Summary of the invention
The present inventor carries out structural modification based on SPR experiment and the integrated use CADD of BIAcore to a kind of propanamide derivative that obtains through primary dcreening operation, gets N-replacement-2-substituted-amino-3-(1H-indol-3-yl) Propionamides compound.The activity (vitro enzyme horizontal checkout) of the inhibition falcipain-2 of test gained compound, the result shows: N-replacement-2-substituted-amino-3-(1H-indol-3-yl) Propionamides compound of the present invention's design, also preparation has stronger falcipain-2 and suppresses active, can be used for preparing the medicine of the malaria that is caused by plasmodium.
The said N-replacement-2-of the present invention substituted-amino-3-(1H-indol-3-yl) Propionamides compound, it has structure shown in the formula I:
Among the formula I: R
1Be hydrogen, C
1-C
6Saturated or the unsaturated alkyl of straight or branched, the C that replaces by the 5-7 unit heterocyclic radical of 5-7 unit virtue (perfume (or spice)) cyclic group, 5-7 unit's heterocyclic radical or the replacement of 5-7 unit virtue (perfume (or spice)) cyclic group, replacement
1-C
6Saturated or the unsaturated alkyl of straight or branched, C
3-C
7Saturated or unsaturated cycloalkyl, 5-7 unit virtue (perfume (or spice)) cyclic group, 5-7 unit virtue (perfume (or spice)) cyclic group of replacement, and close 5-7 unit virtue (perfume (or spice)) cyclic group of 5-7 unit heterocyclic radical, the 5-7 unit heterocyclic radical of 5-7 unit's heterocyclic radical or replacement;
R
2Be ArCH
2-or ArXCO-, wherein: Ar is 5-7 unit virtue (perfume (or spice)) base, and X is O or CH
2
C-2 is configured as R type or S type;
Wherein: substituting group is selected from the 5-7 unit heterocyclic radical of 5-7 unit's virtue (perfume (or spice)) base of described replacement and replacement: halogen (F, Cl, Br or I), C
1-C
4Alkoxyl group, nitro, C
1-C
6The straight or branched alkyl, the C that hydroxyl replaces
1-C
6The straight or branched alkyl, C
3-C
7Saturated or unsaturated cycloalkyl, R
3OCO-, R
3Be C
1-C
6Alkyl, cyano group, amino, trifluoromethyl, trifluoromethoxy, a kind of in sulfydryl or the hydroxyl, two or more; Substituent number is 1-4;
The contained heteroatoms of described heterocyclic radical is N, O and/or S, and contained heteroatomic number is 1-3.
In optimal technical scheme of the present invention, R
1Be C
1-C
3The unsaturated alkyl of straight or branched, the C that replaces by 5 yuan of heterocyclic radicals of phenyl, substituted-phenyl, 5 yuan of heterocyclic radicals or replacement
1-C
3The alkyl of straight or branched, phenyl, substituted-phenyl, and close the phenyl of 5 yuan of heterocyclic radicals, 5 yuan of heterocyclic radicals of 5 yuan of heterocyclic radicals or replacement;
Wherein: substituting group is selected from 5 yuan of heterocyclic radicals of described substituted-phenyl and replacement: halogen, C
1-C
4Alkoxyl group, nitro, the C that hydroxyl replaces
1-C
3The straight or branched alkyl, C
3-C
7Cycloalkyl or R
3OCO-(R
3Be C
1-C
3Alkyl) a kind of in, two or more; Substituent number is 1-4;
The contained heteroatoms of described heterocyclic radical is N, O and/or S, and contained heteroatomic number is 1-3.
Preferred R
1For benzyl, phenyl, to fluorophenyl, to ethoxyl phenenyl, (3 '; 4 '-methylene-dioxy) phenyl, 5 '-cyclopropyl-1 '; 3 ', 4 '-thiadiazoles-2-base, thiophene-2 '-methyl, α-(R)-methylol phenmethyl, propargyl, p-nitrophenyl methyl or to methoxy acyl group phenmethyl.
In another optimal technical scheme of the present invention, R
2Be ArCH
2-or ArXCO-, wherein: Ar is a phenyl, X is O or CH
2
Description of drawings
Fig. 1. positive contrast (Z-Phe-Arg-pNA.HCl) and Compound I
B-1 with the sensing figure of FP-2 protein-interacting
Wherein: the compound concentration that each bar line is represented from top to bottom is followed successively by 1 * 10
-5M, 5 * 10
-6M, 2.5 * 10
-6M, 1.25 * 10
-6M, 6.25 * 10
-7M, 3.125 * 10
-7M and 0.
Fig. 2. be Compound I
B-1, Compound I
B-11 and Compound I
B-12 IC
50The value curve.
Embodiment
In the present invention, described R
3OCO-, its structure such as A formula:
A kind of method for preparing the said N-replacement-2-of the present invention substituted-amino-3-(1H-indol-3-yl) Propionamides compound, its synthesis strategy is as follows:
N-replacement-2 (R, S)-Fang methylamino--3-(1H-indol-3-yl) Propionamides compound I
APreparation:
N-replacement-2-(R, S)-ArX amido-3-(1H-indol-3-yl) Propionamides compound I
BPreparation:
R wherein
1, Ar and X civilian as defined above described.
Specifically comprise the steps:
Compound I
ASynthetic:
1) with N-Boc-D, L-tryptophane, 1-hydroxy benzo triazole, organic bases mix in 0 ℃ with organic solvent and stirred 1 hour, splash into replacement amine, rise to stirring at room 10～48 hours; Use diluted acid, diluted alkaline, washing organic layer respectively, drying, solvent evaporated, the resistates column chromatography for separation, obtain N-replace-2 (R, S)-Boc amino-3-(1H-indol-3-yl) propionic acid amide (compound shown in the formula II is called for short Compound I I, down with);
2) the Compound I I that obtains in the step 1), saturated hydrochloric acid methanol are put in an amount of organic solvent stirring at room 10～24 hours; Steaming desolventizes, obtain N-replace-2 (R, S)-amino-3-(1H-indol-3-yl) propionamide hydrochloride (compound III);
3) with step 2) in the compound III that obtains, add an amount of inorganic alkali lye, use organic solvent extraction, drying, enriched material adds anhydrous magnesium sulfate, methylene dichloride splashes into aromatic aldehyde, 20 ℃-30 ℃ were stirred 5～10 hours; Inorganic alkali lye is washed, the organic layer drying; Solvent evaporated, excess add an amount of methyl alcohol, sodium borohydride, 0 ℃ was stirred 1～5 hour, N-replace-2 (R, S)-Fang methylamino--3-(1H-indol-3-yl) propionic acid amide (Compound I
A).
Compound I
BSynthetic:
Compound III, ArXCOCl and an amount of organic bases are dissolved in the suitable organic solvent, stirred overnight at room temperature, column chromatography, 2 (R, S)-substituted aryl amide group-N-replaces 3-(1H-indoles-3) propionic acid amide (Compound I
B).
N-replacement-2-substituted-amino-3-disclosed in this invention (1H-indol-3-yl) Propionamides compound can be used as the inhibitor of plasmodium falcipain-2, by blocking its catalytic activity, influence the degraded of plasmodium, thereby block plasmodial life cycle host hemoglobin.In addition, it is low and prepare advantages such as simple that compound provided by the present invention also has toxicity, can be used for preparing antimalarial agent.
In following embodiment, will further illustrate the present invention.These embodiment only are used to illustrate the present invention, but do not limit the present invention in any way.All parameters among the embodiment and remaining explanation unless otherwise indicated, all are to be unit with quality (gram).
Embodiment 1
The preparation of N-benzyl-2-(R)-Boc amino-3-(1H-indol-3-yl) propionic acid amide (Compound I I-1)
With 0.62 gram N-Boc-D-tryptophane, 0.42 gram 1-hydroxy benzo triazole, 0.59 gram diisopropylethylamine, 1.27 milliliters of N, the N-diisopropylethylamine mixes stirring 1 hour with 10 milliliters of methylene dichloride in 0 ℃, after splashing into 0.22 milliliter of benzylamine, rise to 20 ℃-30 ℃ and stirred 48 hours; Use diluted acid, diluted alkaline, washing organic layer respectively, drying, solvent evaporated, the resistates column chromatography for separation obtains white solid 0.68 gram, yield 84.4%.mp?142-143℃；
1H?NMR(CDCl
3，500MHz)δ1.39(s，9H)，3.16(m，1H)，3.33(m，1H)，4.26(m，2H)，4.41(m，1H)，6.96(m，3H)，7.13(t，1H)，7.18-7.21(m，4H)，7.35(d，1H)，7.66(d，1H)。
Embodiment 2
The preparation of N-benzyl-2-(R)-amino-3-(1H-indol-3-yl) propionamide hydrochloride (compound III-1)
0.68 gram II-1,5 milliliters of saturated hydrochloric acid methanols are added in 10 milliliters of tetrahydrofuran (THF)s, and 20 ℃ of-30 ℃ of stirrings are spent the night; Steaming desolventizes, and the mixed solution of resistates usefulness minimum of chloroform and methyl alcohol (20/1, V/V) wash, get yellow solid 0.5 gram, yield 87.8%.mp?214-218℃；
1H?NMR(CDCl
3，500MHz)δ3.16(m，1H)，3.25(m，1H)，4.02(s，1H)，4.27(m，2H)，7.00(t，1H)，7.07-7.10(m，3H)，7.19-7.27(m，4H)，7.38(d，1H)，7.66(d，1H)。
Embodiment 3
N-benzyl-2-(R)-aminotoluene base-3-(1H-indol-3-yl) propionic acid amide (Compound I
A-1) preparation
0.25 gram III-1 is dissolved in the ether, adds 10% solution of potassium carbonate, separate ether layer, the water layer ether extraction, the combined ether layer, anhydrous sodium sulfate drying concentrates.Enriched material adds 0.2 gram anhydrous magnesium sulfate, and 5 milliliters of methylene dichloride splash into 0.06 milliliter of phenyl aldehyde under 20 ℃-30 ℃, and 20 ℃-30 ℃ were stirred 6 hours; Saturated sodium carbonate is washed, the organic layer drying; Solvent evaporated, excess add an amount of methyl alcohol, sodium borohydride, and 0 ℃ was stirred 1 hour, got 80 milligrams of yellow solids, yield 34%.mp?38-40℃；
1H?NMR(CDCl
3，500MHz)δ3.03(q，1H)，3.38(q，1H)，3.56-3.60(m，2H)，3.70(d，1H)，4.44(d，2H)，6.95(d，1H)，7.00-7.02(m，2H)，7.11(t，1H)，7.17-7.23(m，6H)，7.26-7.33(m，4H)，7.37(d，1H)；MS(ESI)m/z?406[M+Na]
+。
Embodiment 4
N-benzyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-1) preparation
With 0.15 gram III-1,0.24 milliliter of N, the N-diisopropylethylamine is dissolved in tetrahydrofuran (THF), splashes into benzene oxygen formyl chloride under 20 ℃-30 ℃, and stirring is spent the night, solvent evaporated, and the resistates column chromatography for separation obtains 95 milligrams of white solids, yield 50.3%.mp?176-177℃；
1H?NMR(CDCl
3，500MHz)δ3.14(m，1H)，3.38(m，1H)，4.32(m，2H)，4.48(m，1H)，6.89(m，3H)，7.03(d，2H)，7.08(t，1H)，7.12-7.18(m，6H)，7.29(m，3H)；MS(ESI)m/z?436[M+Na]
+。
Embodiment 5
N-benzyl-2-(S)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-2) preparation
The N-Boc-D-tryptophane is replaced to the N-Boc-L-tryptophane, and all the other desired raw materials, reagent and preparation method get 60 milligrams of white solids, yield 31.7% with embodiment 1,2,4.mp?175-176℃；
1H?NMR(CDCl
3，500MHz)δ3.21(q，1H)，3.46(q，1H)，4.26(q，1H)，4.34(q，1H)，4.55(q，1H)，6.95(s，1H)，6.98(m，2H)，7.10(d，2H)，7.15(t，1H)，7.19-7.26(m，6H)，7.36(q，3H)；MS(ESI)m/z?436[M+Na]
+。
Embodiment 6
N-phenyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-3) preparation
Benzylamine is replaced to aniline, and all the other desired raw materials, reagent and preparation method get 157 milligrams of white solids, yield 86.3% with embodiment 1,2,4.mp?195-197℃；
1H?NMR(CDCl
3，500MHz)δ3.28(q，1H)，3.53(q，1H)，4.71(q，1H)，6.98-7.16(m，6H)，7.21-7.26(t，6H)，7.35-7.42(m，3H)；MS(ESI)m/z?422[M+Na]
+。
Embodiment 7
N-is to fluorophenyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-4) preparation
Benzylamine is replaced to para-fluoroaniline, and all the other desired raw materials, reagent and preparation method get 92 milligrams of yellow solids, yield 48.4% with embodiment 1,2,4.mp?185-189℃；
1H?NMR(CDCl
3，500MHz)δ3.28(q，1H)，3.50(q，1H)，4.70(q，1H)，6.93(t，2H)，7.05-7.17(m，6H)，7.21(m，2H)，7.27(m，1H)，7.35-7.47(m，3H)；MS(ESI)m/z?440[M+Na]
+。
Embodiment 8
N-is to phenelyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-5) preparation
Benzylamine is replaced to p-Phenetidine, and all the other desired raw materials, reagent and preparation method get 72 milligrams of white solids, yield 35.6% with embodiment 1,2,4.mp?180-184℃；
1H?NMR(CDCl
3，500MHz)δ1.39(t，3H)，3.27(q，1H)，3.53(q，1H)，3.98(q，2H)，4.68(q，1H)，6.77(d，2H)，7.08-7.17(m，7H)，7.20-7.25(m，2H)，7.35-7.41(m，3H)；MS(ESI)m/z?466[M+Na]
+。
Embodiment 9
N-[(3 ', 4 '-methylene-dioxy) phenyl]-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-6) preparation
Benzylamine is replaced to 3, the 4-methylene dioxo group aniline, all the other desired raw materials, reagent and preparation method get 52 milligrams of white solids, yield 25.7% with embodiment 1,2,4.mp?186-188℃；
1H?NMR(CDCl
3，500MHz)δ3.26(q，1H)，3.51(q，1H)，4.67(q，1H)，5.93(s，2H)，6.44(d，1H)，6.64(d，1H)，6.92(s，1H)，7.05-7.11(m，3H)，7.13(s，1H)，7.16(t，1H)，7.17-7.24(m，2H)，7.35-7.41(m，3H)；MS(ESI)m/z?466[M+Na]
+。
N-(5 '-cyclopropyl-1 ', 3 ', 4 '-thiadiazoles-2-yl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-7) preparation
Benzylamine is replaced to 5-cyclopropyl-1,3,4-thiadiazoles-2-amine, all the other desired raw materials, reagent and preparation method get 108 milligrams of white solids, yield 52.9% with embodiment 1,2,4.mp?95-101℃；
1H?NMR(CDCl
3，500MHz)δ1.00-1.08(m，2H)，1.12-1.14(m，2H)，2.21(m，1H)，3.38-3.44(m，2H)，4.95(q，1H)，6.97(d，2H)，7.04-7.08(m，2H)，7.15(q，2H)，7.20(d，1H)，7.30(m，3H)；MS(ESI)m/z?470[M+Na]
+。
N-(thiophene-2 '-methyl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-8) preparation
Benzylamine is replaced to thiophene-2-methylamine, and all the other desired raw materials, reagent and preparation method get 100 milligrams of white solids, yield 52.3% with embodiment 1,2,4.mp?184-186℃；
1H?NMR(DMSO-d
6，400MHz)δ2.98(q，1H)，3.16(q，1H)，4.29(q，1H)，4.46(m，2H)，6.91-6.95(m，4H)，6.98(t，1H)，7.06(t，1H)，7.13-7.22(m，2H)，7.29-7.38(m，4H)，7.66(d，1H)；MS(ESI)m/z?442[M+Na]
+。
N-(α-(R)-the methylol phenmethyl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-9) preparation
Benzylamine is replaced to α-(R)-methylol benzene methanamine, and all the other desired raw materials, reagent and preparation method be with embodiment 1,2,4,25 milligrams of oily matter, yield 12.4%.
1H?NMR(DMSO-d
6，500MHz)δ2.98(q，1H)，3.16(q，1H)，4.29(q，1H)，4.46(m，2H)，6.91-6.95(m，4H)，6.98(t，1H)，7.06(t，1H)，7.13-7.22(m，2H)，7.29-7.38(m，4H)，7.66(d，1H)；MS(ESI)m/z?466[M+Na]
+。
Embodiment 13
N-propargyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-10) preparation
Benzylamine is replaced to propargyl amine, and all the other desired raw materials, reagent and preparation method get 87 milligrams of white solids, yield 52.7% with embodiment 1,2,4.mp?173-177℃；
1H?NMR(DMSO-d
6，400MHz)δ2.93-2.99(m，1H)，3.09-3.16(m，2H)，3.88-3.91(m，2H)，4.27(q，1H)，6.92(d，2H)，6.98(t，1H)，7.06(t，1H)，7.13-7.22(m，2H)，7.29-7.35(m，3H)，7.66(d，1H)；MS(ESI)m/z?384[M+Na]
+
Embodiment 14
N-p-nitrophenyl methyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-11) preparation
Benzylamine is replaced to nitro-benzylamine, and all the other desired raw materials, reagent and preparation method get 130 milligrams of white solids, yield 61.9% with embodiment 1,2,4.mp?182-185℃；
1H?NMR(DMSO-d
6，500MHz)δ3.26(q，1H)，3.46(dd，1H)，4.32(dd，1H)，4.42(dd，1H)，4.62(m，1H)，7.01(d，3H)，7.10(d，2H)，7.14-7.24(m，3H)，7.36(t，2H)，7.42(d，1H)，7.73(d，1H)，8.04(d，2H)；MS(ESI)m/z?481[M+Na]
+。
N-is to methoxy acyl group phenmethyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-12) preparation
Benzylamine is replaced to the paraaminomethyl benzoic acid methyl esters, and all the other desired raw materials, reagent and preparation method get 95 milligrams of white solids, yield 44.2% with embodiment 1,2,4.mp?210-213℃；
1H?NMR(DMSO-d
6，500MHz)δ3.45(dd，1H)，3.48(dd，1H)，3.92(s，3H)，4.12(dd，1H)，4.30(dd，1H)，4.37(dd，1H)，4.59(m，1H)，7.06(d，3H)，7.09(d，2H)，7.14-7.24(m，3H)，7.35-7.45(m，3H)，7.75(d，1H)，7.90(d，2H)；MS(ESI)m/z494[M+Na]
+。
Embodiment 16
N-phenmethyl-2-(R)-phenylacetylamino-3-(1H-indol-3-yl) propionic acid amide (Compound I
B-13) preparation
Benzene oxygen chloride of acid is replaced to phenyllacetyl chloride, and all the other desired raw materials, reagent and preparation method get 129 milligrams of yellow solids, yield 68.6% with embodiment 1,2,4.mp?202-205℃；
1H?NMR(CDCl
3，500MHz)δ3.07(q，1H)，3.28(q，1H)，3.54(s，2H)，6.76(s，1H)，6.95(m，2H)，7.13(t，3H)，7.20-7.33(m，7H)，7.36(m，2H)；MS(ESI)m/z?434[M+Na]
+。
Embodiment 17
The compounds of this invention I
B-1 combines determination of activity with Falcipain-2 proteolytic enzyme:
Falcipain-2 proteolytic enzyme and I
B-1 in conjunction with the mensuration of active screening and kinetic constant based on SPR (surface plasma resonance) principle, the instrument of use be Biacore 3000 (Biacore AB, Uppsala, Sweden).
(1) structure of Falcipain-2 plasmid (pQE30-Fal2)
According to Falcipain-2cDNA sequences Design primer, forward and reverse primer are respectively 5 ' CGTGGATCCCAAATGAATTATGAAG3 ' and 5 ' ATATGTCGACTTATTCAATTAATGGAATG3 ', comprise BamH I and Sal I restriction enzyme site, by pcr amplification Falcipain-2 fragment, PCR product after enzyme cut is connected the back and identifies correctly with expression vector pQE30, be transformed into intestinal bacteria M15 (Qiagen) and express.
(2) the proteic expression and purification of Falcipain-2 (FP-2)
The plasmid pQE30-Fal2 that builds changed over to obtain expressing engineering bacteria among the intestinal bacteria M15, engineering bacteria is incubated at overnight incubation (peptone 10g/L in the 10mL LB substratum that contains 100 μ g/mL penbritins and 50 μ g/mL kantlex, yeast extract 5g/L, sodium-chlor 10g/L).Go into 1L by switching in 1: 100 then and contain in the fresh LB substratum of penbritin and kantlex, under 37 ℃, 220 rev/mins of cultivations.When OD600 reaches about 0.8 the time, add IPTG to final concentration 0.5mM, reduce the temperature to 25 ℃ simultaneously and cultivate and carried out induction expression of protein in 12 hours.4000 rev/mins of centrifugal 30 minutes collection thalline are put in-80 ℃ of Ultralow Temperature Freezers preservations and spend the night after collection is good.Thalline is hanged with the buffer 1 of 20mL (and 10mM imidazole, pH 8.0 for 20mM Tris-Cl, 0.5MNaCl), with ultrasonic disruption on the suspension ice bath (300W worked 30 minutes, one time 5 seconds, midfeather 10 seconds).The cell homogenates that obtains after the fragmentation is at 4 ℃, centrifugal 30 minutes with 10000 rev/mins, abandon supernatant, with buffer 2 (6M guanidine HCl 20mM Tris-Cl 250mM NaCl 20mM imidazole, the pH 8.0) dissolution precipitation of 20mL, mild stirring is 1 hour under the room temperature, 10000 rev/mins of centrifugal 30min, and with sample on the supernatant to Bindingbuffer 2 (6M guanidine HCl, 20mM Tris-Cl, 250mM NaCl, pH 8.0) Ni that balance is good
2+On-NTA the post, successively with Wash buffer 1 (8M urea, 20mM Tris-Cl, 500mM NaCl pH 8.0) and the foreign protein of each 30ml flush away non-specific binding of Wash buffer 2 (8M urea, 20mM Tris-Cl, 30mM imidazole), use Elution buffer (8Murea again, 20Mm Tris-Cl, 1M imidazole) 10ml flush away target protein, detect proteic molecular weight and purity with SDS-PAGE.
(3) renaturation of FP-2 inclusion body protein
The albumen that purifying is obtained adds 10mM DTT, 37 ℃ down temperature bathe after 45 minutes, protein solution is diluted to the 10 μ g/ml (dialyzate: 100mM Tris-Cl of dialysing, 1mM EDTA, 20%glycerol, 250mM L-arginine, 1mMGSH, 1mM SSG, pH 8.0) spend the night.The albumen that dialysis is good concentrates the mensuration that promptly can be used as enzyme inhibition activity.
(4) the proteic coupling of FP-2
After thoroughly cleaning Biacore 3000 machines, steady to baseline with HBS-EP damping fluid (3mMEDTA and 0.005% (v/v) surfactant P20, pH 7.4 for 10mM Hepes, 150mM NaCl) balancing machine.0.2MN-ethyl-N '-dimethylaminopropyl carbodiimide (N-ethyl-N '-dimethyl aminopropyl carbodiimide) and 50mMMN-N-Hydroxysuccinimide (EDC/NHS) 1: 1 are mixed, with 5 μ L/min sample introductions 7 minutes with the activation chip surface.FP-2 albumen 10mM sodium acetate, pH4.2, being diluted to final concentration is 69 μ g/ml, with 5 μ L/min flow velocity sample introductions.At last, use the 1M diethanolamine hydrochloride, pH 8.5 is with 5 μ L/min flow velocity sample introductions 7 minutes, the sealing chip surface, and the final proteic coupling amount of FP-2 is about 9300RU.
(5) screening compound
Substrate Z-Phe-Arg-pNAHCl (Bachem AG) is as positive control.I
B-1 usefulness 100%DMSO dissolving, mother liquid concentration is 10mM.With HBS-EP damping fluid diluted compounds, to final concentration be 1 μ M and 10 μ M, the final concentration of DMSO is 0.1%.According to I
B-1 with chip on the proteic bonded RU of FP-2 (Response Unit, resonance units) value, judge whether compound has in conjunction with activity.Have in conjunction with active compound and can carry out detailed dynamic experiment.The result proves I
B-1 has tangible the combination with FP-2 albumen.
(6) kinetic determination
I
B-1 usefulness work damping fluid HBS-EP (containing 0.1%DMSO) is made into different concentration gradients respectively, and with 30 μ l/min sample introduction 1min, the 2min that dissociates stablizes 2min with same buffer then.Obtain I
B-1 with the sensing figure of FP-2 protein-interacting, use 1: 1 (Langmuir) combination model or steady-state model in the Biacore analysis software to carry out match again, obtain definite kinetics and thermodynamic equilibrium constant.
(6) test-results: see Table 1. positive controls and I
B-1 with the test result of FP-2 protein binding constant
Table 1.
Embodiment 18
The compounds of this invention suppresses active mensuration to falcipain-2 proteolytic enzyme percentage
(1) renaturation of proteic expression and purification of Falcipain-2 (FP-2) and FP-2 inclusion body protein
Referring to embodiment 16
(2) The compounds of this invention is to the mensuration of FP-2 enzyme inhibition activity
100mM NaOAc at 197 μ L, 10mM DTT, add FP-2 albumen (final concentration 10 μ g/ml) in the buffer system of pH 5.5 and be dissolved in the testing compound solution of DMSO, final concentration 10 μ M and 0 μ M (negative control) are hatched under the room temperature behind the 30min with MD SpectraMax M5 microplate reader in excitation 355nm; Emission 460nm place surveys the RFU value in the 15min continuously, calculates speed of reaction K
m, draw testing compound percent inhibition under 10 μ M with following formula,
Calculation formula is:
(control group K
mValue-experimental group K
mValue)/control group K
mValue * 100%
(3) the compound activity test result sees Table 2.N-replacement-2-substituted-amino-3-(1H-indol-3-yl) Propionamides compound to falcipain-2 inhibiting rate data.
Table 2.
Embodiment 19
Part of compounds of the present invention is to falcipain-2 proteolytic enzyme half effective inhibition concentration (IC
50) mensuration
Choose 10 μ M inhibiting rates and survey IC at the compound more than 30%
50, select the suitable compound concentration gradient, experimental technique and system such as embodiment 17.According to the compound speed of reaction K that the FP-2 enzyme is lived under different concns
m, the inhibiting rate that the computerized compound is lived to the FP-2 enzyme under different concns uses the Sigmoidal formula to carry out the IC that match obtains compound with origin software
50Value, (N-replacement-2-substituted-amino-3-(1H-indol-3-yl) Propionamides compound is lived to the falcipain-2 enzyme and is suppressed IC to the results are shown in Table 3
50) and Fig. 2.
Table 3.
As from the foregoing: N-replacement-2-substituted-amino-3-of the present invention (1H-indol-3-yl) Propionamides compound has all shown positive findings at falcipain-2 proteolytic enzyme in conjunction with test and enzyme inhibition test, should demonstrate,prove it and have inhibition falcipain-2 pharmacological action.
Claims (5)
1. a structure is preparing the application for the treatment of in the medicine that is caused malaria by plasmodium suc as formula the Propionamides compound shown in the I:
Among the formula I: R
1Be C
1-C
6The unsaturated alkyl of straight or branched, the C that replaces by the 5-7 unit aromatic ring yl of 5-7 unit aromatic ring yl, replacement
1-C
6The saturated alkyl of straight or branched, 5-7 unit aromatic ring yl, 5-7 unit's aromatic ring yl of replacement or and close the 5-7 unit aromatic ring yl of 5-7 unit heterocyclic radical;
R
2Be ArCH
2-or ArX (CO)-, wherein: Ar is a 5-7 unit aromatic ring yl, and X is O or CH
2
C-2 is configured as R type or S type;
Wherein: the substituting group of the 5-7 unit aromatic ring yl of described replacement is selected from: halogen, C
1-C
4Alkoxyl group, nitro, C
1-C
6The straight or branched alkyl, the C that hydroxyl replaces
1-C
6The straight or branched alkyl, R
3O (CO)-, R
3Be C
1-C
6Alkyl, cyano group, amino, trifluoromethyl, trifluoromethoxy, in sulfydryl or the hydroxyl one or two or more kinds; Substituent number is 1-4;
The contained heteroatoms of described heterocyclic radical is N, O and/or S, and contained heteroatomic number is 1-3.
2. application as claimed in claim 1 is characterized in that, wherein R
1Be C
1-C
3The unsaturated alkyl of straight or branched is by the C of phenyl or substituted-phenyl replacement
1-C
3The alkyl of straight or branched, phenyl, substituted-phenyl or and close the phenyl of 5 yuan of heterocyclic radicals;
Wherein: the substituting group of described substituted-phenyl is selected from: halogen, C
1-C
4Alkoxyl group, nitro, the C that hydroxyl replaces
1-C
3Straight or branched alkyl or R
3Among the OCO-one or two or more kinds; R
3Be C
1-C
3Alkyl.
3. application as claimed in claim 1 is characterized in that, wherein R
1For benzyl, phenyl, to fluorophenyl, to ethoxyl phenenyl, (3 ', 4 '-methylene-dioxy) phenyl, α-(R)-methylol phenmethyl, propargyl, p-nitrophenyl methyl or to methoxy acyl group phenmethyl.
4. application as claimed in claim 1 is characterized in that, wherein R
2Be ArCH
2-or ArX (CO)-, wherein: Ar is a phenyl, X is O or CH
2
5. a Propionamides compound is preparing the application for the treatment of in the medicine that is caused malaria by plasmodium;
Wherein said Propionamides compound is N-benzyl-2-(R)-aminotoluene base-3-(1H-indol-3-yl) propionic acid amide; N-benzyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-benzyl-2-(S)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-phenyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-is to fluorophenyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-is to phenelyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-[(3 '; 4 '-methylene-dioxy) phenyl]-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-(5 '-cyclopropyl-1 '; 3 ', 4 '-thiadiazoles-2-yl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-(thiophene-2 '-methyl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-(α-(R)-the methylol phenmethyl)-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-propargyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-p-nitrophenyl methyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide; N-is to methoxy acyl group phenmethyl-2-(R)-benzene oxygen formamido group-3-(1H-indol-3-yl) propionic acid amide or N-phenmethyl-2-(R)-phenylacetylamino-3-(1H-indol-3-yl) propionic acid amide.
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