CN103965066A - Double-target inhibitor for leukotriene A4 hydrolase and cyclooxygenase as well as purpose thereof - Google Patents
Double-target inhibitor for leukotriene A4 hydrolase and cyclooxygenase as well as purpose thereof Download PDFInfo
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- 0 *c(cc1C(O)=O)ccc1O Chemical compound *c(cc1C(O)=O)ccc1O 0.000 description 1
- VRNPOZHMXLXTEB-UHFFFAOYSA-N COc(c(C(OCCCc1ccccc1)=O)c1)ccc1F Chemical compound COc(c(C(OCCCc1ccccc1)=O)c1)ccc1F VRNPOZHMXLXTEB-UHFFFAOYSA-N 0.000 description 1
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- PJSWZGHHUVINHR-UHFFFAOYSA-N OCCCc(cc1)ccc1F Chemical compound OCCCc(cc1)ccc1F PJSWZGHHUVINHR-UHFFFAOYSA-N 0.000 description 1
- ADQJEIMFMFIJKB-UHFFFAOYSA-N Oc(c(C(OCCCc1ccccc1)=O)c1)ccc1F Chemical compound Oc(c(C(OCCCc1ccccc1)=O)c1)ccc1F ADQJEIMFMFIJKB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a double-target inhibitor for leukotriene A4 hydrolase and cyclooxygenase as well as a purpose of the double-target inhibitor. The double-target inhibitor is a compound shown in a general formula (I), wherein X1 and X2 are respectively and independently separated into hydrogen, halogen, alkyl or alkoxy; Y represents hydrogen, hydroxyl, halogen or alkyl; Z represents substituent groups at the fourth position and/or the fifth position of a benzoyl core benzene ring, and respectively represents hydrogen, halogen, amino, alkyl acylamino, alkyl substituted amino, trifluoromethyl or carboxyl alkyl acylamino; n is 2 to 4. The compound can be used for preparing medicine for treating, preventing or inhibiting inflammation such as arthritis and rheumatoid arthritis.
Description
Technical field
The present invention relates to treatment and prevent the medicine of various inflammation, particularly dual-target inhibitor of a class leukotriene A 4 hydrolase and cyclooxygenase and uses thereof, belongs to multifunctional drug design field.
Background technology
Inflammation is to have the defensive raction that the biological tissue of vascular system occurs damage factor, when this defensive raction excessive activation, out of hand in, inflammatory factor will be attacked damage human body autologous tissue, diseases induced, may threat to life when serious.The generation of inflammation is the complex process that a polymolecular participates in regulation and control.Therefore, a lot of people recognize: in the early stage of medicament research and development, should consider the effect of selected drug targets in disease overall network, utilize the means of systems biology to carry out medicament research and development.This new medicinal design theory is at present in the exploratory stage.
Contriver place seminar explored how for the molecular network of complex disease, to carry out modeling, dynamic analysis and dosage regimen prediction since 2004, proposed to find for disease-related molecular network the method for calculation of optimum control scheme, and searching molecular network is changed the method for calculation MTOI of optimal solution to standard state by morbid state.Contriver utilizes MTOI to study arachidonic acid (AA) metabolism network relevant to mankind's inflammation, point out in this network is changed to standard state by morbid state, when being necessary to carry out a plurality of target spot, control, a kind of inhibitor that this controls a plurality of target spots roughly the same time, is called many targets inhibitor.
Arachidonic acid metabolism network is the network that produces inflammatory factor, Phospholipase A2 in this metabolism network (PLA2), cyclooxygenase (COX), 5-lipoxygenase (5-LOX), leukotriene A 4 hydrolase (LTA4H) etc., it is respectively the emphasis target of anti-inflammatory drug design, the highly selective list target inhibitor obtaining based on these drone designs is widely used, and wherein a part has been given play to significant curative effect in clinical.In AA metabolism network, phosphatide is discharged arachidonic acid by PLA2 hydrolysis, subsequently by two metabolic pathway: (one) generates various prostaglandin(PG)s (PGs) via COX path; (2) via LOX path, generate leukotriene (LTs), lipid peroxide.Research shows, between these two paths, exists and influences each other, and path of single inhibition will cause another path inflammatory Cytokines Expression amount to improve.Many targets inhibitor that design acts in two paths in this network simultaneously can have following advantage:
One, suppress inflammatory factor leukotriene and the prostaglandin(PG) that two pathways metabolisms produce simultaneously, increase antiphlogistic effects, avoided causing because of the inhibition of a pathways metabolism situation of the activation of another pathways metabolism.
Two, many targets inhibitor is little compared with single target inhibitor to the inhibition of single enzyme, also less (the dual-target inhibitor Da Bufeilong for example of caused side effect, inhibition, with marketed drug indomethacin is suitable, has been proved to be and has not had stomach toxicity).
Summary of the invention
The dual-target inhibitor that the object of this invention is to provide leukotriene A 4 hydrolase and cyclooxygenase, comprises the preparation method of this class inhibitor, is used for the treatment of and prevents various inflammation.
We will combine and set up a set of effective many target agents research strategy FdenoD from head design method (Drug de novo Design) with medicine based on fragment drug research strategy (Fragment Based Drug Design, FBDD).This ability, in COX-2 and the design of LTA4H difunctional inhibitor, is successfully obtained to COX-2 and the LTA4H difunctional inhibitor of a series of novel structures.
First, according to 3 rule requests, taking into full account on the basis of existing structure activity study, by single depressant of functions of existing COX and LTA4H be split as molecular weight 300 with interior sheet segment molecule, by business buy and simply organic synthesis strategy set up one by 21 molecular micro chip phase libraries of fragment.Then, under higher concentration, in (0.5～1mM) test piece phase library, molecule is active to the inhibition of COX-2 and LTA4H, obtains 6 and has the active guide's fragment of difunctional inhibition.Then, use AutoDock4.0 by difunctional fragment molecular docking to two target, choose the higher reasonable conformation of a plurality of marking as seed conformation, use LigBuilder3.0 to derive design.In the design of the first round, controlling institute increases molecular weight in 50, from give a mark rank select 6 exemplary configuration in 1000 design molecule and synthesize and determination of activity.At this, take turns and in optimization, obtain 1 difunctional bioactive molecule and carry out follow-up second and take turns LigBuilder3.0 design.In the second design result of taking turns, extract public skeleton 2-hydroxyl-5-benzaminic acid-3-phenyl propyl ester (PKUMDL_AAD_101) and synthesize and activity rating.Active result shows that PKUMDL_AAD_101 has higher COX-2 and the difunctional inhibition of LTA4H is active.Analyze PKUMDL_AAD_101 and target binding pattern, as shown in Figure 1, polar group on benzoyl core phenyl ring has crucial effect in two targets, as amino, form hydrogen bond with the Glu296 of LTA4H and the Tyr355 of COX-2, the Arg563 of hydroxyl and LTA4H and the Ser530 of COX-2 form hydrogen bond; The introducing of another phenyl ring hydrophobic grouping contributes to the binding ability of raising and two targets.Based on these structural informations, we design and have synthesized a series of 2-hydroxyl-5-benzaminic acid-3-phenyl propyl esters and analogue (PKUMDL_AAD_102-224) thereof.
The dual-target inhibitor general structure specific as follows of leukotriene A 4 hydrolase provided by the invention and cyclooxygenase:
General formula (I)
In general formula (I), X
1represent hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxyl group; X
2represent hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxyl group; Y represents hydrogen, hydroxyl, halogen, C1-C4 alkyl; Z representative is positioned at the substituting group on 4 and/or 5 of benzoyl core phenyl ring, can be identical or different, and the hydrogen of respectively doing for oneself, halogen, amino, C1-C6 alkylamidoalkyl, C1-C6 alkyl substituted amido, trifluoromethyl or C1-C6 carboxyalkyl amide group; N=2～4.
Described C1-C4 alkyl is methyl, ethyl, propyl group for example; Described C1-C4 alkoxyl group is methoxyl group, oxyethyl group for example; Described C1-C6 alkylamidoalkyl (RCONH-, wherein R is C1-C5 alkyl) is acetamido, propionamido-for example; Described C1-C6 alkyl substituted amido is methylamino, dimethylin for example; Described C1-C6 carboxyalkyl amide group is carboxylic propyl amides base, carboxylic butyl amide base for example.
General formula of the present invention (I) compound can be prepared by following method: with substituted benzoic acid, react with the primary alconol of end containing the 2-4 carbochain of substituted-phenyl, make substituted benzoyl acid alkyl ester.Further, can carry out hydro-reduction to substituted benzoyl acid alkyl ester, methylate, the reaction such as amidation, obtain corresponding compound.For example:
With the phenylformic acid of 4 and/or 5 nitro replacements, react with the primary alconol of end containing the 2-4 carbochain of substituted-phenyl, first prepare the phenylformic acid ester that nitro replaces, by hydro-reduction, react the target product PKUMDL_AAD that obtains amino-contained, again the PKUMDL_AAD of amino-contained is methylated and/or amidation, corresponding obtaining containing methylamino, amide group PKUMDL_AAD compound.
This synthetic route chemical substance used is all commercially available product, while reacting, the working method adopting and operation steps and reaction conditions and intermediate etc., it is all the methodology of organic synthesis design of knowing according to those skilled in the art, implement, and be disclosed in each embodiment.
Synthetic method of the present invention has following outstanding feature:
1. raw material is easy to get, cheap.
2. experiment is simple, and aftertreatment is relatively easy.
Experimental period short, be easy to amplification quantity synthetic.
The synthetic compound of the present invention is as the difunctional inhibitor of leukotriene A 4 hydrolase and cyclooxygenase, and live test and whole blood test result of its enzyme show, synthesized compound shows activity.
The pharmacologically acceptable salt of the compounds of this invention is also in protection scope of the present invention.The pharmacologically acceptable salt of described the compounds of this invention refers to the salt of apparent the compounds of this invention to those skilled in the art, for salt nontoxic and that can advantageously realize the pharmacokinetic property of the compounds of this invention, those compounds of favourable pharmacokinetic properties such as absorption, distribution, metabolism and excretion are provided, consider raw materials cost, water absorbability, stability, the easy practical factor such as crystallinity, yield, bulk drug mobility simultaneously.The acid of using while preparing pharmacologically acceptable salt or alkali can be used conventional organic acid or mineral acid and common alkali.
The invention still further relates to described compound and pharmaceutical salts thereof the purposes in the pharmaceutical compositions of the patient's condition by leukotriene A 4 hydrolase and cyclooxygenase mediation for the preparation for the treatment of, prevention or inhibition experimenter, particularly for the preparation for the treatment of, prevention or suppress the purposes in experimenter's the pharmaceutical compositions of inflammation, such as can be applicable in the pharmaceutical compositions for the treatment of of arthritis, rheumatoid arthritis etc.Compound of the present invention also can be applicable to for the preparation of suppressing in the pharmaceutical compositions of leukotriene A 4 hydrolase and cyclooxygenase enzymic activity.Described pharmaceutical compositions comprises at least one the compounds of this invention being dispersed in pharmaceutically acceptable carrier.The amount of the compounds of this invention in pharmaceutical compositions is enough to suppress the activity of leukotriene A 4 hydrolase and cyclooxygenase.
The leukotriene A 4 hydrolase that synthetic method of the present invention makes and the difunctional inhibitor of cyclooxygenase can be used separately or add conventional pharmaceutical excipient to make the uses such as tablet, suppository, capsule or injection liquid.The example of solid excipient comprises the various materials that are usually used in pill and tablet, as lactose, starch, methylcellulose gum etc.The example of liquid oral vehicle comprises ethanol, glycerine, water etc.
Accompanying drawing explanation
Fig. 1 is the binding pattern schematic diagram of PKUMDL_AAD_101 and target, wherein: a) PKUMDL_AAD_101 and LTA4H binding pattern schematic diagram; B) PKUMDL_AAD_101 and COX-2 binding pattern schematic diagram.
Embodiment
Following examples are used for illustrating the present invention, represent to put into practice method of the present invention, and it is without any restrictions to scope of the present invention.Those skilled in the art may find and apparently for them realize additive method of the present invention, all should think that those methods are included in the scope of the present invention.
General experimental technique:
In the GCD-500G high-purity hydrogen generator of hydrogenation employing Beijing Jia Wei Kechuang Science and Technology Ltd. and BLT-2000, press hydrogenation instrument.Nmr spectrum data (
1h-NMR and
13c-NMR) by U.S. Varian Mercury300M and BruckerARX400spectrometer, recorded.Use deuterated methanol or contain tetramethylsilane be interior target deuterochloroform, deuterated dimethyl sulfoxide as solvent, coupling constant is usingd Hz as unit, abbreviation used is illustrated as: s=is unimodal, d=is bimodal, t=triplet, q=quartet, m=multiplet, the mono-broad peak of br=.High resolution mass spectrum data (HRMS) are recorded by U.S.'s Brsucker ApexIV FTICRMS instrument.Fusing point shows that by Tyke, Beijing X-4 of Instrument Ltd. numeral micro melting point apparatus records.
Methylene dichloride is through CaH
2non-aqueous processing; Sodium Metal 99.5 and benzophenone Non-aqueous processing for tetrahydrofuran (THF) and toluene.Other common solvent are the analytical reagent of direct purchase.Silica gel that column chromatography for separation is used is that (200-300 order) bought from Chinese Haiyang Chemical Plant, Qingdao, and the sherwood oil boiling point using is 60-90 ℃.Thin-layer chromatography chromatosheet is the efficient plate of GF254, from Chinese Haiyang Chemical Plant, Qingdao, buys.
Synthesizing of embodiment 12-hydroxyl-5-benzaminic acid-3-phenyl propyl ester (PKUMDL_AAD_101)
(1) intermediate 11 is synthetic
In being equipped with the 25mL round-bottomed flask of drying tube, adding and contain 5-NITROSALICYLIC ACID (0.37g, 2.0mmol), 3-phenylpropyl alcohol (0.27g, 2.0mmol), the THF solution 10mL of EDCI (0.46g, 2.4mmol) and catalytic amount DMAP, stirred overnight at room temperature.Decompression steams THF, and with acetic acid ethyl dissolution, washing, saturated salt washing, anhydrous sodium sulfate drying, cross silicagel column and obtain product 11 (0.43g, productive rate 72%).
(2) target compound PKUMDL_AAD_101's is synthetic
Ethyl acetate solution 20mL containing compound 11 (0.1g, 0.33mmol) is proceeded to hydrogenation instrument reaction tubes, add Pd/C (0.02g).Install reaction tubes, regulating hydrogenation instrument pressure is 0.4MPa, replaces after 3 air, starts reaction.After about 2h, reaction finishes, and with diatomite filtration under diminished pressure, removes palladium carbon, and Rotary Evaporators steams solvent, obtains brown oil PKUMDL_AAD_101 (0.086g, productive rate 95%).
1H-NMR(400MHz，CDCl
3)：δ10.26(s，1H)，7.30(m，2H)，7.21(m，3H)，7.07(d，1H)，6.87(dd，1H)，6.82(d，1H)，4.34(t，2H)，3.44(br，2H)，2.79(t，2H)，2.11(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ170.0，154.9，141.0，138.2，128.5(2)，128.4(2)，126.1，124.3，118.2，114.7，112.3，64.6，32.3，30.0；HRMS(ESI)：calcd for C
16H
18NO
3[(M+H)
+]272.1281，found272.1280.
Target compound PKUMDL_AAD_102 to PKUMDL_AAD_105 obtains with above-mentioned same method is synthetic.
3-benzaminic acid-3-phenyl propyl ester (PKUMDL_AAD_102): faint yellow oily matter, productive rate 70%.
1H-NMR(400MHz，CDCl
3)：δ7.42(d，1H)，7.30(m，3H)，7.21(m，4H)，6.85(dd，1H)，4.31(t，2H)，3.78(br，2H)，2.78(t，2H)，2.09(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ166.8，146.5，141.3，131.4，129.3，128.5(4)，126.0，119.7，119.4，115.7，64.2，32.3，30.3；HRMS(ESI)：calcd for C
16H
18NO
2[M+H
+]256.1332，found256.1332.
2-methyl-5-benzaminic acid-3-phenyl propyl ester (PKUMDL_AAD_103): faint yellow oily matter, productive rate 65%.
1H-NMR(400MHz，CDCl
3)：δ7.29(t，2H)，7.20(m，3H)，7.01(d，1H)，6.72(dd，1H)，4.29(t，2H)，3.60(br，2H)，2.77(t，2H)，2.47(s，3H)，2.07(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ167.9，144.2，141.3，132.6，130.3，129.7，128.5(4)，126.1，119.0，116.9，64.2，32.5，30.4，20.9；HRMS(ESI)：calcd for C
17H
20NO
2[M+H
+]270.1489，found270.1490.
2-hydroxyl-5-benzaminic acid-2-phenyl chlorocarbonate (PKUMDL_AAD_104): faint yellow solid, productive rate 57%.
1H-NMR(400MHz，CDCl
3)：δ10.18(s，1H)，7.33(t，2H)，7.26(m，3H)，7.10(d，1H)，6.86(dd，1H)，6.81(d，1H)，4.53(t，2H)，3.38(br，2H)，3.07(t，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.8，154.9，138.3，137.5，128.9(2)，128.6(2)，126.8，124.3，118.2，114.7，112.3，65.7，35.1；HRMS(ESI)：calcd for C
15H
16NO
3[M+H
+]258.1125，found258.1125；m.p.83℃。
2-hydroxyl-5-benzaminic acid-4-butyloxy phenyl (PKUMDL_AAD_105): faint yellow oily matter, productive rate 52%.
1H-NMR(400MHz，CDCl
3)：δ10.26(s，1H)，7.29(m，2H)，7.20(m，3H)，7.13(d，1H)，6.87(dd，1H)，6.82(d，1H)，4.34(t，2H)，3.43(br，2H)，2.69(t，2H)，1.80(m，4H)；
13C-NMR(100MHz，CDCl
3)：δ170.0，154.9，141.9，138.2，128.4(4)，125.9，124.3，118.2，114.7，112.4，65.1，35.4，28.2，27.7；HRMS(ESI)：calcd for C
17H
29NO
3[M+H
+]286.1438，found286.1440.
Synthesizing of embodiment 22-hydroxyl-5-fluorine phenylformic acid-3-phenyl propyl ester (PKUMDL_AAD_106)
(1) 2-methoxyl group-5-fluorophenyl carbamate is synthetic
The DMF40mL reaction system tube sealing that is dissolved with 5-fluorosalicylic acid (0.6g, 4mmol), methyl iodide (2.4g, 16mmol) and salt of wormwood (2.2g, 16mmol), spends the night 100 ℃ of reactions.Cooling reaction system, uses saturated ammonium chloride solution cancellation, ethyl acetate dilution.Organic phase is washed successively, saturated salt washing, anhydrous sodium sulfate drying, and decompression steams solvent and obtains product 2-methoxyl group-5-fluorophenyl carbamate (0.7g, productive rate 99%).R
f=0.5 (silica gel, ethyl acetate: sherwood oil=1: 5 (volume ratios));
1h-NMR (300MHz, CDCl
3): δ 7.5 (m, 1H), 7.17 (m, 1H), 6.91 (dd, 1H), 3.88 (d, 6H);
(2) 2-methoxyl group-5-fluorobenzoic acid is synthetic
2-methoxyl group-5-fluorophenyl carbamate is joined in 10% potassium hydroxide/methyl alcohol (1: 2) mixing solutions to stirring at room reaction.LTC monitoring, after reacting completely, steams part methyl alcohol, with 2M salt acid for adjusting pH to 2～3, separates out solid.Filter, be drying to obtain product 2-methoxyl group-5-fluorobenzoic acid (0.61g, productive rate 96%).
(3) intermediate 13 is synthetic
Be equipped with in the 25mL round-bottomed flask of drying tube and add and contain 2-methoxyl group-5-fluorobenzoic acid (0.17g, 1.0mmol), compound 12 (0.13g, 0.91mmol), the THF solution 10mL of EDCI (0.21g, 11mmol) and catalytic amount DMAP, stirred overnight at room temperature.Decompression steams THF, and with acetic acid ethyl dissolution, washing, saturated salt washing, anhydrous sodium sulfate drying, cross silicagel column and obtain product 13 (0.10g, productive rate 40%).
1H-NMR(300MHz，CDCl
3)：δ7.45(dd，1H)，7.32～7.14(m，6H)，6.94(dd，1H)，4.32(t，2H)，3.89(s，3H)，2.79(t，2H)，2.08(m，2H)；
(4) 2-hydroxyl-5-fluorine phenylformic acid-3-phenyl propyl ester (PKUMDL_AAD_106) is synthetic
-40 ℃, under argon shield, in the 5mL dichloromethane solution that contains 13 (0.033g, 0.11mmol), slowly add BBr3 (0.05mL, 0.53mmol).Finish and close cooling system, make it from-40 ℃, rise to gradually room temperature.After TLC detection reaction is complete, by a small amount of methyl alcohol cancellation system.Add 40mL ethyl acetate, water, salt water washing successively, anhydrous sodium sulfate drying.Removal of solvent under reduced pressure obtains faint yellow oily matter PKUMDL_AAD_106 (0.030g, productive rate 91%).
1H-NMR(400MHz，CDCl
3)：δ10.57(s，1H)，7.44(dd，1H)，7.30(m，2H)，7.19(m，4H)，6.93(dd，1H)，4.37(t，2H)，2.79(t，2H)，2.13(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.3(d)，157.9(d)，155.1(d)，140.8，128.6(2)，123.4(2)，126.2，123.2(d)，118.8(d)，115.0(d)，112.4(d)，65.1，32.2，30.0.
Target compound PKUMDL_AAD_107～PKUMDL_AAD_111 obtains with above-mentioned same method is synthetic.
2-hydroxyl-5-fluorine phenylformic acid-2-phenyl chlorocarbonate (PKUMDL_AAD_107): faint yellow oily matter, overall yield 28%.
1H-NMR(400MHz，CDCl3)：δ10.43(s，1H)，7.38(dd，1H)，7.29(m，5H)，7.10(m，1H)，6.85(dd，1H)，4.48(t，2H)，3.01(t，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.2(d)，157.9(d)，155.1(d)，137.2，128.9(2)，128.7(2)，126.9，123.2(d)，118.8(d)，115.0(d)，112.4(d)，66.1，35.0.
2-hydroxyl-5-fluorine phenylformic acid-4-butyloxy phenyl (PKUMDL_AAD_108): faint yellow oily matter, overall yield 30%.
1H-NMR(400MHz，CDCl
3)：δ10.58(s，1H)，7.48(dd，1H)，7.28(m，2H)，7.17(m，4H)，6.94(dd，1H)，4.36(t，2H)，2.69(t，2H)，1.80(m，4H)；
13C-NMR(100MHz，CDCl
3)：δ169.3(d)，157.9(d)，155.1(d)，141.7，128.4(4)，126.0，123.2(d)，118.8(d)，115.0(d)，112.4(d)，65.6，35.4，28.1，27.6.
2-hydroxyl-5-trifluoromethylbenzoic acid-3-phenyl propyl ester (PKUMDL_AAD_109): faint yellow oily matter, overall yield 25%.
1H-NMR(400MHz，CDCl
3)：δ11.15(s，1H)，8.05(d，1H)，7.68(dd，1H)，7.29(m，2H)，7.20(m，3H)，7.07(d，1H)，4.41(t，2H)，2.79(t，2H)，2.16(m，2H)；
2-hydroxyl-5-trifluoromethylbenzoic acid-2-phenyl chlorocarbonate (PKUMDL_AAD_110): faint yellow oily matter, overall yield 36%.
1H-NMR(400MHz，CDCl
3)：δ11.05(s，1H)，8.08(d，1H)，7.67(dd，1H)，7.26(m，5H)，7.06(d，1H)，4.58(t，2H)，3.11(t，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.1，164.0，137.1，132.1(q)，128.9(2)，128.7(2)，127.6(q)，126.9，123.8(q)，121.6(q)，118.4，112.4，66.4，35.0.
2-hydroxyl-5-trifluoromethylbenzoic acid-4-butyloxy phenyl (PKUMDL_AAD_111): faint yellow oily matter, overall yield 21%.
1H-NMR(400MHz，CDCl
3)：δ11.16(s，1H)，8.09(d，1H)，7.67(dd，1H)，7.30(m，2H)，7.20(m，3H)，7.07(d，1H)，4.40(t，2H)，2.71(t，2H)，1.81(m，4H)；
13C-NMR(100MHz，CDCl
3)：δ168.3，163.0，140.6，131.1(q)，127.4(4)，126.5(q)，125.0，122.8(q)，120.6(q)，117.4，111.4，64.9，34.3，27.0，26.5.
Synthesizing of embodiment 32-hydroxyl-5-acetylaminohydroxyphenylarsonic acid phenylformic acid-3-phenyl propyl ester (PKUMDL_AAD_112)
(1) intermediate 15 is synthetic
Be equipped with in the 50mL round-bottomed flask of drying tube and add and contain 2-methoxyl group-5-nitrobenzoic acid (0.39g, 2.0mmol), compound 12 (0.27g, 2.0mmol), the THF solution 20mL of EDCI (0.42g, 2.2mmol) and catalytic amount DMAP, stirred overnight at room temperature.Decompression steams THF, and with acetic acid ethyl dissolution, washing, saturated salt washing, anhydrous sodium sulfate drying, cross silicagel column and obtain product 14 (0.40g, productive rate 63%) (containing part unreacted compound 12 completely), directly drops into next step.
Ethyl acetate solution 50mL containing compound 14 (0.40g, 1.3mmol) is proceeded to hydrogenation instrument reaction tubes, add Pd/C (0.08g).Install reaction tubes, regulating hydrogenation instrument pressure is 0.4MPa, replaces after 3 air, starts reaction.After about 2h, reaction finishes, and with diatomite filtration under diminished pressure, removes palladium carbon, and Rotary Evaporators steams solvent, obtains amine intermediate, directly drops into acetylization reaction.
Above-mentioned amine intermediate is dissolved in 20mL THF, under stirring, adds Acetyl Chloride 98Min. (142 μ L, 2mmol) and triethylamine (0.44mL, 3.2mmol).After room temperature reaction 6h, concentrate system, by the 30mL shrend system of going out, ethyl acetate extraction (30mL * 3).Merge organic phase, the pickling of 1M salt, washing, salt washing, anhydrous sodium sulfate drying.Steam solvent, by ethyl acetate, sherwood oil system, cross silicagel column and obtain pure compound 15 (0.22g, three-step reaction amounts to productive rate 34%).
(2) target compound PKUMDL_AAD_112's is synthetic
BBr
3demethyl method, with embodiment 2, obtains faint yellow oily matter PKUMDL_AAD_112, productive rate 53%.
1H-NMR(400MHz，CDCl
3)：δ10.65(s，1H)，7.85(d，1H)，7.57(dd，1H)，7.30(t，2H)，7.20(t，4H)，6.94(d，1H)，4.35(t，2H)，2.78(t，2H)，2.16(s，3H)，2.12(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.7，168.4，158.6，140.9，129.4，129.0，128.5(4)，126.1，121.6，118.0，112.2，64.9，32.2，29.9，24.2；HRMS(ESI)：calcd for C
18H
20NO
4[M+H
+]314.1387，found314.1390.
Target compound PKUMDL_AAD_113～PKUMDL_AAD_114 obtains with above-mentioned same method is synthetic.
2-hydroxyl-5-acetaminobenzoic acid-2-phenyl chlorocarbonate (PKUMDL_AAD_113): faint yellow oily matter, overall yield 20%.
1H-NMR(400MHz，CDCl
3)：δ10.60(s，1H)，7.96(d，1H)，7.52(dd，1H)，7.34(m，2H)，7.27(m，4H)，6.93(d，1H)，4.53(t，2H)，3.08(t，2H)，2.16(s，3H)；
13C-NMR(100MHz，CDCl
3)：δ169.6，168.3，158.6，137.3，129.4，129.0(2)，128.9，128.7(2)，126.8，112.7，118.0，112.2，66.0，35.0，24.3；HRMS(ESI)：calcd for C
17H
18NO
4[M+H
+]300.1230，found300.1234.
2-hydroxyl-5-acetylamino benzoic acid-4-butyloxy phenyl (PKUMDL_AAD_114): faint yellow oily matter, overall yield 14%.
1H-NMR(400MHz，CDCl
3)：δ10.67(s，1H)，7.90(d，1H)，7.56(dd，1H)，7.29(t，2H)，7.19(t，4H)，6.94(d，1H)，4.35(t，2H)，2.69(t，2H)，2.15(s，3H)，1.80(m，4H)；
13C-NMR(100MHz，CDCl
3)：δ169.8，168.3，158.6，141.8，129.4，129.0，128.4(4)，125.9，121.6，118.0，112.3，65.4，35.4，28.1，27.6，24.2；HRMS(ESI)：calcd for C
19H
22NO
4[M+H
+]328.1543，found328.1547.
Synthesizing of embodiment 42-hydroxyl-5-carboxylic propyl amides yl benzoic acid-3-phenyl propyl ester (PKUMDL_AAD_115)
Under argon shield, in 2mL pyridine/methylene dichloride (1: the 1) mixing solutions that contains Succinic anhydried (0.025g, 0.25mmol), add compound PKUMDL_AAD_101 (0.20g, 0.75mmol).After stirring at room 16h, solvent evaporated, obtains Off-white solid PKUMDL_AAD_115 (0.043g, productive rate 46%) with ether crystallization.
1H-NMR(400MHz，DMSO)：δ12.12(s，1H)，10.29(s，1H)，9.96(s，1H)，8.13(d，1H)，7.69(dd，1H)，7.28(m，4H)，7.19(m，1H)，6.94(d，1H)，4.30(t，2H)，2.75(t，2H)，2.53(br，4H)，2.04(m，2H)；
13C-NMR(100MHz，DMSO)：δ173.8，169.8，168.7，155.9，140.9，131.3，128.3(4)，127.1，125.9，119.7，117.4，112.4，64.4，31.3，30.8，29.6，28.8；HRMS(ESI)：calcd for C
20H
22NO
6[M+H
+]372.1442，found372.1444；m.p.153℃。
Target compound PKUMDL_AAD_116 obtains with above-mentioned same method is synthetic.
2-hydroxyl-5-carboxylic butyl amide yl benzoic acid-3-phenyl propyl ester (PKUMDL_AAD_116): productive rate 39%.
1H-NMR(400MHz，CDCl
3)：δ10.66(s，1H)，7.90(d，1H)，7.55(dd，1H)，7.49(br，1H)，7.29(m，2H)，7.19(m，4H)，6.92(d，1H)，4.34(t，2H)，2.77(t，2H)，2.45(m，4H)，2.08(m，4H)；
13C-NMR(100MHz，CDCl
3)：δ170.7，169.7，158.6，140.9，129.3，128.9，128.5(4)，126.1，121.5，118.0，112.2，64.9，35.9，32.9，32.2，29.9，20.5；HRMS(ESI)：calcd for C
21H
24NO
6[M+H
+]386.1598，found386.1600.
Synthesizing of embodiment 52-fluoro-5-benzaminic acid-3-phenyl propyl ester (PKUMDL_AAD_117) and 2-fluoro-5-methylamino acid-3-phenyl propyl ester (PKUMDL_AAD_118)
The synthetic of compound 16 obtained by 2-fluorine-5-nitro benzoic acid and hydrocinnamyl alcohol esterification, and method is shown in that PKUMDL_AAD_101 is synthetic.Methanol solution 15mL containing compound 16 (0.078g, 0.26mmol) is proceeded to hydrogenation instrument reaction tubes, add Pd/C (0.016g).Install reaction tubes, regulating hydrogenation instrument pressure is 0.4MPa, replaces after 3 air, starts reaction.After about 4h, reaction finishes, and with diatomite filtration under diminished pressure, removes palladium carbon, and Rotary Evaporators steams solvent, crosses silicagel column obtain brown color oily matter PKUMDL_AAD_1180.015g, productive rate 20%, R by ethyl acetate, sherwood oil system
f=0.4 (silica gel, ethyl acetate: sherwood oil=1: 5)
1h-NMR (400MHz, CDCl
3): δ 7.29 (t, 2H), 7.20 (q, 3H), 7.08 (q, 1H), 6.97 (t, 1H), 6.72 (m, 1H), 4.33 (t, 2H), 3.74 (br, 1H), 2.84 (s, 3H), 2.79 (t, 2H), 2.09 (m, 2H);
13c-NMR (100MHz, CDCl
3): δ 165.2,154.7 (d), 145.4 (d), 141.3,128.5 (4), 126.0,118.8 (d), 118.0 (d), 117.4 (d), 113.9,64.4,32.2,31.1,30.3; HRMS (ESI): calcd forC
17h
19fNO
2[M+H
+] 288.1394, found288.1396.; Obtain brown color oily matter PKUMDL_AAD_117 (0.032g, productive rate 45%) simultaneously.R
f=0.2 (silica gel, ethyl acetate: sherwood oil=1: 5)
1h-NMR (400MHz, CDCl
3): δ 7.28 (t, 2H), 7.20 (m, 3H), 7.15 (q, 1H), 6.94 (q, 1H), 6.79 (m, 1H), 4.33 (t, 2H), 3.65 (br, 2H), 2.79 (t, 2H), 2.08 (m, 2H);
13c-NMR (100MHz, CDCl
3): δ 164.8,155.4 (d), 142.3,141.2,128.5 (4), 126.0,120.6 (d), 118.9 (d), 117.5 (d), 117.1,64.5,32.2,30.2; HRMS (ESI): calcd for C
16h
17fNO
2[M+H
+] 274.1238, found274.1243.
Target compound PKUMDL_AAD_119-120 obtains with above-mentioned same method is synthetic.
2-hydroxyl-4-methylamino phenylformic acid-3-phenyl propyl ester (PKUMDL_AAD_119): brown color solid, productive rate 24%.
1H-NMR(400MHz，CDCl
3)：δ11.07(s，1H)，7.59(d，1H)，7.29(m，2H)，7.20(m，3H)，6.08(m，2H)，4.29(t，2H)，4.23(br，1H)，2.86(s，3H)，2.77(t，2H)，2.08(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ170.2，163.9，155.3，141.2，131.0，128.5(d，4)，126.0，105.4，101.7，97.2，63.7，32.3，30.3，30.0；HRMS(ESI)：calcd for C
17H
20NO
3[M+H
+]286.1438，found286.1440；m.p.85℃。
2-hydroxy-4-aminobenzoic acid-3-phenyl propyl ester (PKUMDL_AAD_120): faint yellow solid, productive rate 50%.
1H-NMR(400MHz，CDCl
3)：δ10.99(s，1H)，7.60(d，1H)，7.29(t，2H)，7.20(m，3H)，6.14(t，2H)，4.29(t，2H)，4.09(br，2H)，2.77(t，2H)，2.08(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ170.1，163.7，153.3，141.1，131.6，128.5(d，4)，126.1，106.8，103.2，100.8，63.9，32.2，30.2；HRMS(ESI)：calcdfor C
16H
18NO
3[M+H
+]272.1281，found272.1281；m.p.78℃。
Synthesizing of embodiment 62-hydroxyl-5-benzaminic acid-3-(4-fluorophenyl) propyl ester (PKUMDL_AAD_121)
(1) microcosmic salt is synthetic
In the 50mL round-bottomed flask that contains 10mL sewage toluene, add triphenyl phosphorus (1.31g, 5.0mmol) and methyl bromoacetate (0.76g, 5.0mmol), prolong and drying tube are installed, reflux is spent the night.Cooling reaction system, removes by filter toluene, obtains microcosmic salt white solid, and vacuum-drying obtains product (1.9g, productive rate 92%).
(2) synthesizing fluoro cinnamic acid methyl esters
Under dry Ar protection, microcosmic salt (1.08g, 2.6mmol) is joined in the 15mL anhydrous THF solution that is equipped with potassium tert.-butoxide (0.27g, 2.4mmol), finish and stir 1.5h.Then splash into the 5mL THF solution that contains p-Fluorobenzenecarboxaldehyde (0.25g, 2.0mmol), stirring and refluxing 12h.Reaction solution cool to room temperature, pours in 30mL water, by extracted with diethyl ether (40mL * 3 time).Merging organic phase water, saturated NaCl wash successively, anhydrous sodium sulfate drying.Except ethyl acetate, sherwood oil for desolventizing, cross silicagel column and obtain product to fluoro cinnamic acid methyl esters (0.32g, productive rate 89%).
(3) intermediate 17 is synthetic
Contain the 20mL anhydrous methanol of fluoro cinnamic acid methyl esters (0.32g, 1.8mmol) and palladium carbon (0.06g) is packed in hydrogenation instrument, adjusting hydrogen pressure is 0.4MPa, replaces after three gases, reacts.Stopped reaction after 8h, crosses diatomite, after decompression steams solvent, will obtain product hydrogenation to fluoro cinnamic acid methyl esters, without processing, directly drops into next step.
At 0 ℃, above-mentioned hydrogenation is slowly added drop-wise in the ether suspension of LiAlH4 (0.075g, 12.0mmol) the 5mL anhydrous ether solution of fluoro cinnamic acid methyl esters.Finish and remove ice bath, stirring at room to reaction finishes (TLC detects, and approximately needs 2 hours).After reaction finishes, add successively 0.75mL water, 2.2mL15%NaOH solution, 2.2mL water, 3.7g anhydrous sodium sulphate, stirs after 10min, filters and collects filtrate, with ether washing leaching cake repeatedly.Combined ether layer, anhydrous sodium sulfate drying, solvent evaporated obtains product alcohol (0.25g, two-step reaction amounts to productive rate 90%).
(4) PKUMDL_AAD_121's is synthetic
The esterification of alcohol 17 and 5-NITROSALICYLIC ACID and follow-up hydrogenating reduction are all identical with PKUMDL_AAD_101 synthetic method, obtain yellow solid PKUMDL_AAD_121.Productive rate 51%.
1H-NMR(400MHz，CDCl
3)：δ10.23(s，1H)，7.16(q，2H)，7.07(d，1H)，6.99(t，2H)，6.88(dd，1H)，6.83(d，1H)，4.33(t，2H)，3.43(br，2H)，2.76(t，2H)，2.08(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ168.9，160.4(d)，154.0，137.2，135.5(d)，128.8，128.7，123.4，117.2，114.4，114.1，113.6，111.3，63.4，30.5，29.2；HRMS(ESI)：calcd forC
16H
17FNO
3[M+H
+]290.1187，found290.1185；m.p.64℃。
Target compound PKUMDL_AAD_122-124 obtains with above-mentioned same method is synthetic.
2-hydroxyl-5-benzaminic acid-3-(4-chloro-phenyl-) propyl ester (PKUMDL_AAD_122): brown color oily matter, productive rate 42%.
1H-NMR(400MHz，CDCl
3)：δ10.25(s，1H)，7.30(m，2H)，7.18(m，3H)，7.06(dd，1H)，6.87(dd，1H)，6.82(d，1H)，4.33(t，2H)，3.45(br，2H)，2.79(t，2H)，2.11(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ170.0，155.0，141.0，138.2，128.5(d，4)，126.1，124.3，118.2，114.7，112.3，64.6，32.3，30.0；HRMS(ESI)：calcd for C
16H
17ClNO
3[M+H
+]306.0891，found306.0893.
2-hydroxyl-5-benzaminic acid-3-(4-p-methoxy-phenyl) propyl ester (PKUMDL_AAD_123): brown color oily matter, productive rate 55%.
1H-NMR(400MHz，CDCl
3)：δ10.26(s，1H)，7.13(d，2H)，7.04(d，1H)，6.85(m，4H)，4.33(t，2H)，3.79(s，3H)，3.37(br，2H)，2.73(t，2H)，2.06(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ170.0，158.0，155.0，138.3，133.1，129.4(2)，124.3，118.2，114.7，114.0(2)，112.4，64.6，55.3，31.4，30.2；HRMS(ESI)：calcd for C
17H
20NO
4[M+H
+]302.1387，found302.1389.
2-hydroxyl-5-benzaminic acid-3-(the fluoro-4-chloro-phenyl-of 3-) propyl ester (PKUMDL_AAD_124): faint yellow oily matter, productive rate 48%.
1H-NMR(400MHz，CDCl
3)：δ10.24(s，1H)，7.26(m，1H)，7.07(dd，1H)，6.99(dd，1H)，6.89(m，2H)，6.82(d，1H)，4.33(t，2H)，3.45(t，2H)，2.78(t，2H)，2.10(m，2H)；
13C-NMR(100MHz，CDCl
3)：δ169.9，163.0(d)，154.9，143.5(d)，138.3，129.9(d)，124.4，124.1(d)，118.2，115.3(d)，114.6，113.0(d)，112.2，64.4，32.1，29.8；HRMS(ESI)：calcdfor C
16H
16ClFNO
3[M+H
+]324.0797，found324.0801.
The pure enzyme inhibition activity of embodiment 7LTA4H is measured
(1) solution allocation
EIA Buffer:10mL10 * liquid storage+90mL Mili-Q ultrapure water.
Wash Buffer: with ultrapure water with the dilution proportion concentrated solution of 1: 400 (volume ratio), and add Tween-20 to concentration be 0.5ml/L.Tween-20 need pipette with syringe.
LTB
4in AChE Tracer:100dtn pressed powder, add 6mL EIABuffer and 60 μ L red pigments.4 ℃ of Refrigerator stores, available in 4 weeks.
LTB
4in Antiser um:100dtn pressed powder, add 6mL EIABuffer and 60 μ L blue pigmentss.4 ℃ of Refrigerator stores, available in 4 weeks.
LTB
4antiser Wash Buffer: according to Mili-Q water dilution Wash Buffer liquid storage for 1: 400 (volume ratio) ratio, add Tween-20 to concentration be 0.5mL/L.Note, pipettes Tween-20 with syringe.
In Ellman ' s Reagent:100dtn pressed powder, add 20mL Mili-Q water.Matching while using, keeps in Dark Place.
(2) sample preparation
Substrate: get-80 ℃ of LTA
4liquid storage, dilutes 500 times, adds 20 μ L in system, and reaction system substrate final concentration is 150nM.
Reaction buffer: 10mM NaH
2pO
4/ Na
2hPO
4, pH7.4,2～5mg/mL BSA.
Testing compound: be dissolved in DMSO and be made into 20 * liquid storage, add 20 μ L in system.DMSO concentration in system is controlled in 10%.
Enzyme: 1OD
280lTA
450 times of H dilutions, add 20 μ L in system, reaction system enzyme final concentration is 19nM.
While preparing testing sample, first by testing compound, enzyme in reaction buffer at 37 ℃ of preincubate 10min, then add substrate reactions 10min.Then get 100 times of termination reactions of EIABuffer dilution for 10 μ L reaction solutions.
(3) standard configuration
Get 100 μ LLTB
4standard to one clean 1.5ml pipe, with 900 μ L ultrapure water dilutions, vibration extremely mixes completely, now LTB in solution
4concentration is 5ng/ml.Then be diluted in EIABuffer in certain sequence.
(4) experimental procedure
In 96 orifice plates, add 150 μ L reaction Buffer, 20 μ LLTA
4h, 10 μ L micromolecular compounds are at 37 ℃ of jolting 10min; Add 20 μ LLTA
4, 37 ℃ of jolting reaction 10min.By carry out cancellation reaction by EIABuffer diluted system (20 times).With EIA Buffer, the system after cancellation being diluted to different multiples afterwards does sample and determines final extension rate (1～5 times of general cancellation system dilution can be at LTB
4in ELISA measurement range).Under application of sample process is shown in:
After finishing, reaction measures according to EIA test kit described method.
(5) data processing
Inhibiting rate %=[1-(I-B)/(I
0-B)] * 100%, IC
50by Origin8.0, use Hill1 matching to obtain.Test result is in Table 1.
The pure enzyme inhibition activity of embodiment 8COX is measured
(1) reagent preparation
Survey the Buffer:100mM PBS that lives, pH6.5,1mg/mL Gelatin
Gelatin: purchased from Beijing Si Baihui biotechnology limited liability company 10% aqueous solution (100mg/mL)
COX2 (ovine) and COX1 (ovine): all purchased from Cayman company, with surveying the Buffer that lives, dilute rear use, to reacting first speed in 20～50mOD/min (deduction back end).
TMPD:N, N, N, N-tetramethyl-p-phenylenylendiamine hydrochloride, purchased from Sigma Aldrich company, is made into 100mM liquid storage with Mili-Q water, divides and installs to 200 μ LEP pipes ,-80 ℃ of preservations.During use, with Mili-Q water, be diluted to 1mM again, matching while using.In surveying live body system, ultimate density is 100 μ M.
Geuapol X-100: with Buffer dilution, being made into concentration is 44mM solution.Surveying live body is that final concentration is 2mM.
AA substrate: be 100mM purchased from Cayman stock concentrations, be diluted to 1mM during use with Mili-Q water, reaction system final concentration is 100 μ M.In 30 minutes, use, can the proper extension time in ice bath.
Heme: according to providing Heme to be configured use in the COX Inhibitor Screening Assay Kit of Cayman company (560131) or ColorimetricCOX (ovine) Inhibitor Screening Assay Kit (760111).In 560131: 10 μ L+240 μ L Buffer; In 760111: 88 μ LHeme+1.912mL Buffer.Diluent room temperature can be stablized 12 hours.
Compound: be dissolved in and be made into 50 * solution in DMSO.
(2) experimental procedure
146 μ L Buffer+10 μ LHeme+10 μ LCOX/Buffer (jolting 1min makes Heme and the abundant combination of COX)+10 μ LGenapol+4 μ L compound/DMSO (jolting 15min)+20 μ LTMPD+20 μ LAA initiate reaction assay 610nm dynamics data, get the first speed of front 40s and calculate.Application of sample process sees the following form:
(3) data processing
Inhibiting rate %=[1-(I-B)/(I
0-B)] * 100%, IC
50by Origin8.0, use Hill1 matching to obtain.Test result is in Table 1.
Embodiment 9PGE
2whole blood assay
Equipment and reagent:
Heparin tube: non-(Vacuette) disposal vacuum heparin tube (containing Lithium heparinate anti-freezing), specification, the 6mL/9mL of replacing that adopts that company of Austrian Gray (Greiner Bio-One GmbH) to produce.
LPS:LPS is purchased from Sigma-Aldrich company.Be dissolved in PBS solution and be made into the liquid storage that contains 40mM PBS, 100mM NaCl, 5mg/mL LPS, divide and install to 200 μ L EP pipes in-80 ℃ of Refrigerator stores.
Inhibitor: be dissolved in DMSO.COX measures inhibitor liquid storage and is 100 *, in blood, final DMSO content is in 1%.
Experimental procedure:
Day1, configuration inhibitor 100 * liquid storage; Be ready to take out heparin tube used, syringe needle and insulated tank; Get 1 μ L testing inhibitor and be added to round bottom 96 orifice plate bottoms, in a hole, add therein 1 μ LDMSO as full reference alive.
Day2, the blood of extraction respective volume, divides and is added in above-mentioned 96 orifice plates after mixing, the fresh blood that every hole adds 100 μ L to contain antithrombotics.37 ℃ add 2 μ LLPS after hatching 15～20min, hatch 24h for 37 ℃.
Day3, is imbedded in 5～10min in ice bath and reacts with cancellation hatching 96 orifice plates after 24h.After go in 200 μ L EP pipes, 3000rpm, 4 ℃, centrifugal 5min, taking out 20 μ L supernatants, to be saved to-80 ℃ of refrigerators to be measured.
By positive reference and back end (DMSO) sample PGE
2eIA damping fluid dilutes different multiples, upper PGE
2elisa plate carries out sample mensuration, so that determine should extension rate.Note, generally dilutes 100,200,500 3 concentration gradients and can determine.
Day4, determines that according to preliminary experiment extension rate carries out PGE to whole samples
2eLISA measures.Test result is in Table 1.
Embodiment 10LTB
4whole blood assay
Adopt calcium ion carrier A 23187 to stimulate the 5-LOX path of AA metabolism network.Use LTB
4the principal product LTB of EIA kit measurement 5-LOX path
4evaluate tested molecule to inflammatory mediator LTB
4inhibition ability.In this method, a reaction molecular is to LTB
4the impact generating, therefore to 5-LOX and LTA
4h whole blood characterizes can use this method.
Instrument and reagent:
Heparin tube: non-(Vacuette) disposal vacuum heparin tube (containing Lithium heparinate anti-freezing), the specification: 6mL/9mL replaced that adopts that company of Austrian Gray (Greiner Bio-One GmbH) to produce.
A23187:A23187 is purchased from Sigma-Aldrich company.Be dissolved in DMSO, being configured to concentration is 12.5mM solution, 4 ℃ of preservations.
Inhibitor: be dissolved in DMSO.LTA
4h measures inhibitor liquid storage and is 200 *.In blood, final DMSO content is in 1%.
Experimental procedure:
Day1, configuration inhibitor 200 * liquid storage; Be ready to take out heparin tube used, syringe needle and insulated tank;
Get 1 μ L testing inhibitor and be added to round bottom 96 orifice plate bottoms, in a hole, add therein 1 μ LDMSO as full reference alive.
Day2, the blood of extraction respective volume, divides and is added in above-mentioned 96 orifice plates after mixing, the fresh blood that every hole adds 200 μ L to contain antithrombotics.37 ℃ add 1 μ LA23187 to continue to hatch 30min at 37 ℃ after hatching 15～20min.
Ice bath buries 5～10min cancellation reaction, goes in 200 μ L EP pipes, and 3000rpm, 4 ℃, centrifugal 5min, it is to be measured that taking-up 50 μ L supernatants are saved to-80 ℃ of refrigerators.
By positive reference and back end (DMSO) sample PGE
2eIA damping fluid dilutes different multiples, upper PGE
2elisa plate carries out sample mensuration, so that determine should extension rate.Note, generally dilutes 100,200,500 3 concentration gradients and can determine.
Day3, determines that according to preliminary experiment extension rate carries out PGE to whole samples
2eLISA measures.Test result is in Table 1.
Using the COX-2 positive is to evaluate with reference to flurbiprofen (Flurbiprofen) and LTA4H inhibitor Ref-1 to above-mentioned survey live body.
Table 1. has the test result of active target compound
From the vitro enzyme test result alive of table 1, can find out that compound PKUMDL_AAD_101 and PKUMDL_AAD_122 can suppress LTA simultaneously
4h and COX; In the experiment of people's whole blood, above-claimed cpd is for LTA
4the inhibition IC of H and COX
50reach micromole or sub-micro mole number magnitude (PKUMDL_AAD_101COX-2IC
50=18.0 μ M, LTA
4h IC
50=12.6 μ M; PKUMDL_AAD_104COX-2IC
50=7.9 μ M, LTA4H IC
50=8.1 μ M; PKUMDL_AAD_105COX-2IC
50=13.0 μ M, LTA
4h IC
50=5.4 μ M; PKUMDL_AAD_121COX-2IC
50=20.1 μ M, LTA
4h IC
50=11.8 μ M; PKUMDL_AAD_122COX-2IC
50=7.0 μ M, LTA4H IC
50=7.1 μ M; PKUMDL_AAD_123COX-2IC
50=13.7 μ M, LTA
4h IC
50=8.9 μ M; PKUMDL_AAD_124COX-2IC
50=15.9 μ M, LTA
4h IC
50=6.2 μ M), wherein live in vitro PKUMDL_AAD_122 all to show stronger inhibition in test and the experiment of people's whole blood active by enzyme, and COX-2/COX-1 selectivity is 11.1, approaching with theoretical prediction optimum value 7, belonging to low selective COX-2-2 inhibitor, is potential LTA
4many targets of H/COX inhibitor.
Claims (8)
1. compound shown in general formula (I):
General formula (I)
In general formula (I), X
1represent hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxyl group; X
2represent hydrogen, halogen, C1-C4 alkyl or C1-C4 alkoxyl group; Y represents hydrogen, hydroxyl, halogen, C1-C4 alkyl; Z representative is positioned at the substituting group on 4 and/or 5 of benzoyl core phenyl ring, can be identical or different, and the hydrogen of respectively doing for oneself, halogen, amino, C1-C6 alkylamidoalkyl, C1-C6 alkyl substituted amido, trifluoromethyl or C1-C6 carboxyalkyl amide group; N=2～4.
2. compound shown in general formula as claimed in claim 1 (I), is characterized in that X
1represent hydrogen, halogen, methyl or methoxy; X
2represent hydrogen or halogen C1-C4 alkyl; Y represents hydrogen, hydroxyl or halogen; Z representative is positioned at the identical or different substituting group on 4 and/or 5 of carboxylic acid phenyl ring, is hydrogen, halogen, amido, acetamido, methylamino, trifluoromethyl carboxylic propyl amides base or carboxylic butyl amide base.
3. compound shown in general formula as claimed in claim 1 (I), is characterized in that, this compound is one of following compounds PKUMDL_AAD_101 to PKUMDL_AAD_124:
4. the preparation method of compound shown in general formula (I) described in a claim 1, substituted benzoic acid reacts with the primary alconol of end containing the 2-4 carbochain of substituted-phenyl, make substituted benzoyl acid alkyl ester, or further substituted benzoyl acid alkyl ester is carried out hydro-reduction, methylated and/or amidate action, obtain corresponding compound.
5. preparation method as claimed in claim 4, it is characterized in that, with the phenylformic acid of 4 and/or 5 nitro replacements, react with the primary alconol of end containing the 2-4 carbochain of substituted-phenyl, first make the phenylformic acid ester that nitro replaces, then by hydro-reduction, react the target product PKUMDL_AAD that obtains amino-contained:
Again the PKUMDL_AAD of amino-contained is methylated and/or amidation, corresponding obtaining containing methylamino and/or amide group PKUMDL_AAD compound.
Described in claim 1 compound shown in general formula (I) as the purposes of the dual-target inhibitor of leukotriene A 4 hydrolase and cyclooxygenase.
Described in claim 1 compound shown in general formula (I) and pharmaceutical salts thereof for the preparation for the treatment of, prevention or suppress the purposes in the medicine of the patient's condition that mediated by leukotriene A 4 hydrolase and cyclooxygenase.
8. purposes as claimed in claim 7, is characterized in that, the described patient's condition by leukotriene A 4 hydrolase and cyclooxygenase mediation refers to inflammation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111620942A (en) * | 2020-06-12 | 2020-09-04 | 中国科学院昆明动物研究所 | Mature peptide of batus brucei leukotriene A4hydrolase inhibitor Motisin and application thereof |
| CN112979513A (en) * | 2021-02-07 | 2021-06-18 | 武汉工程大学 | Chiral sulfoxide containing styrene monomer and preparation method thereof |
| CN113735721A (en) * | 2021-10-09 | 2021-12-03 | 上海昕凯医药科技有限公司 | Method for synthesizing 3-ethylamino-4-methylphenol |
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2013
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111620942A (en) * | 2020-06-12 | 2020-09-04 | 中国科学院昆明动物研究所 | Mature peptide of batus brucei leukotriene A4hydrolase inhibitor Motisin and application thereof |
| CN111620942B (en) * | 2020-06-12 | 2021-10-15 | 中国科学院昆明动物研究所 | Mature peptide of batus brucei leukotriene A4hydrolase inhibitor Motisin and application thereof |
| CN112979513A (en) * | 2021-02-07 | 2021-06-18 | 武汉工程大学 | Chiral sulfoxide containing styrene monomer and preparation method thereof |
| CN113735721A (en) * | 2021-10-09 | 2021-12-03 | 上海昕凯医药科技有限公司 | Method for synthesizing 3-ethylamino-4-methylphenol |
| CN113735721B (en) * | 2021-10-09 | 2023-09-29 | 上海昕凯医药科技有限公司 | Method for synthesizing 3-ethylamino-4-methylphenol |
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