CN107434819A

CN107434819A - Indoles TEMPO conjugates and its purposes being far apart in protection in organ confrontation ischemical reperfusion injury

Info

Publication number: CN107434819A
Application number: CN201710079866.XA
Authority: CN
Inventors: 毕伟; 毕悦
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2017-02-15
Filing date: 2017-02-15
Publication date: 2017-12-05
Anticipated expiration: 2037-02-15
Also published as: CN107434819B

Abstract

The purposes being far apart the invention discloses the indoles TEMPO conjugates with general formula I and its in protection in organ confrontation ischemical reperfusion injury.Research shows that the indoles TEMPO conjugates for a series of novel that the present invention synthesizes show antiinflammatory property in the dropsy of ear mouse model that dimethylbenzene induces, and then demonstrates levels of reactive oxygen species excess generation and mitochondria dysfunction that these compounds can protect cells from the I/R inductions of simulation.In addition, present invention has demonstrated that indoles TEMPO conjugates can mitigate the multiple organ injury induced due to intestines I/R damages in rodent, and the pharmacological characteristic and mechanism of action of these indoles TEMPO conjugates are related to converging action, including reducing ability caused by free radical by lipid peroxidation, and with the decrease acted on the ROS mediated activations in inflammatory process.The present invention provides a kind of new technological means for anti-intestine ischemia/reperfusion injury.

Description

Indoles-TEMPO conjugates and its protection be far apart organ confrontation ischemical reperfusion injury In purposes

Technical field

The present invention relates to a kind of indoles-TEMPO conjugates and application thereof, more particularly to indoles-TEMPO conjugates are being protected The purposes that shield is far apart in organ confrontation ischemical reperfusion injury, the invention belongs to pharmaceutical technology field.

Background technology

Intestine ischemia/Reperfu- sion (I/R) damage represents the clinical syndrome occurred in various environment, including abdominal aneurvsm, Small intestine transplantation, strangulated hernia and neonatal necrotizing enterocolitis.The Reperfu- sion induction multiform of superior mesenteric artery (SMA) The activation of core neutrophil leucocyte (PMN), along with the release of proinflammatory substance and the generation of more polyradical.Including hydroxyl free These groups including base, by promoting lipid and DNA oxidations/peroxidating, increase the formation of free radical and related mitochondria Depolarising, and final Apoptosis, played a role in the Pathological Physiology of intestines I/R damages.In addition, these I/R are damaged The proinflammatory factor activated after wound circulates in vein and lymphatic system, induces long-range organ damage, facilitate systemic inflammatory response and ARDS, this is added in the research of multiple intervening measure beneficial effects that have recorded and mitigate ROS influences To emphasize.

4- hydroxyls-TEMPO (TEMPOL) is a kind of piperidines nitrogen oxides of stabilization, as in ESR spectrum Water-soluble intermediate be widely used.TEMPOL can permeate biomembrane, be accumulated in cytosol, can remove super oxygen in vitro Thing anion, therefore may act as " SOD (superoxide dismutase) analogies ".In addition, TEMPOL may be by removing super oxygen Thing anion, or pass through intracellular Fe²⁺Concentration is reduced to reduce the formation of hydroxyl radical free radical.Latter procedure passes through Fenton Or the formation of Haber-Weiss response inhabitation hydroxyl radical free radicals.With SOD, catalase or other external sources are applied anti-oxidant Agent is different, and the readily permeable cell membrane of nitrogen oxides simultaneously passes through blood-brain barrier.Therefore, nitrogen oxides is for related to oxidative stress Disease and damage with unique treatment potentiality.

Being related to indole derivatives has anti-oxidant and antiinflammatory property report.In this respect, there is harmine (harmane), harmaline (harmaline) and Harmalol (harmalol) are by limiting hyaluronic acid, chondrin Former and immunoglobulin G oxidative damage suppresses the lipid peroxidation in liver.Other as shown by data, β-carboline alkaloid lead to Cross removing ROS and play protectiveness antioxidation；Our laboratory be recently developed it is a series of show enhancing it is anti-oxidant With the indoles beta-carboline derivatives of anti-inflammatory activity.Due to ROS and inflammatory leukocytes risen in the pathogenesis that I/R is damaged it is important Neutrophil cell infiltration is with treatment correlation almost certainly after effect, antioxidant therapy and suppression ischemic.In many In the case of when considering therapeutic scheme, the increase that combined therapy is generally improved by symptom improves therapeutic efficiency.Due to different Pharmacokinetic profile, the Formulations for systemic administration of two kinds of potential synergy medicines will lead to therapeutic target with different efficiency.However, When co-application medicament introduces as single chemical entities, it is possible to achieve the advantage in treatment.Accordingly we assume that simultaneously according to this after Continuous research：This part of radicals scavenging characteristic of TEMPO, observed plus the indole derivatives with exploitation before us Anti-inflammatory activity, the new conjugate with the anti-oxidant of collaboration enhancing, anti-inflammatory and anti-ischemic activity will be produced.In order to assess this It is individual it is assumed that we are prepared for a series of new indoles-TEMPO conjugates, and characterize them and experimental in vitro and in vivo lack Weaken the biological capacity of I/R damages in blood/re-perfusion model.

The content of the invention

The technical problems to be solved by the invention are to provide a series of new indoles-TEMPO conjugates, and provide these The purposes that conjugate is far apart in organ confrontation ischemical reperfusion injury in protection.

In order to achieve the above object, present invention employs following technological means：

Inventor has synthesized the indoles-TEMPO conjugates of a series of novel, and they induce ear water in dimethylbenzene Antiinflammatory property is shown in swollen mouse model, and then demonstrate these compounds to protect cells from the I/R inductions of simulation ROS (levels of reactive oxygen species) excess generations and mitochondria dysfunction.In addition, present invention has demonstrated that indoles-TEMPO sews Compound can mitigate the multiple organ injury induced due to intestines I/R damages in rodent, and these indoles-TEMPO is conjugated The pharmacological characteristic and mechanism of action of thing are related to converging action, including reduce ability caused by free radical by lipid peroxidation, And with the decrease acted on the ROS mediated activations in inflammatory process.

Specifically, a kind of indoles-TEMPO conjugates of the present invention, it has the structure shown in formula I：

Wherein, X is carbon, oxygen, nitrogen, sulphur, silicon, phosphorus or boron atom；X is combined by saturation or unsaturated bond；X can be with appointing What substituent forms key；N is selected from 4-6 integer；

Wherein, R is selected from the C of hydrogen, substituted or unsubstituted, straight chain, ring-type or side chain_1-5Alkyl or carbonyl, amide groups or The cycloalkyl that substituted amide groups or R collectively form the cycloalkyl of nitrogen atom or are substituted with N atoms.

In the present invention, it is preferred to, in formula I,Group is selected from

Wherein R₁-R₄Selected from hydrogen, substituted or unsubstituted, straight chain, the C of ring-type or side chain_1-5Alkyl.

In the present invention, it is preferred to, described substituted or unsubstituted, straight chain, the C of ring-type or side chain_1-5Alkyl or carbonyl Base includes the C substituted through amino, 3- nitros guanidine radicals, imidazole radicals, phenyl, hydroxy phenyl, alkylthio group, hydroxyl or indyl_1-5Alkane Base or carbonyl, described substituted amide groups include 1- epoxides -2,2,6,6- tetramethyl piperidine -4- bases) -1,2,3-triazoles -4- The formamido or acetamido of ylmethyl substitution.

In the present invention, it is preferred to, R is selected from hydrogen, methyl, isopropyl, normal-butyl -2- bases, isobutyl group, 2- amino -2- oxygen For ethyl, imidazol-4 yl-methyl, benzyl, 4- aminobutyls, para hydroxybenzene methyl, 2-methylmercaptoethyl, methylol, Yin Diindyl -3- bases-methyl, R1 and N atoms be collectively forming pyrroles -2, N- base, 1- hydroxyethyls,3- amino 3- oxopropyls

In a particular embodiment of the present invention, described indoles-TEMPO conjugates are selected from the chemical combination with following structure Thing：

Further, the invention also provides the method for the described indoles-TEMPO conjugates of synthesis, including following step Suddenly：

Since optical activity L-Trp, parent B-carboline 1 is prepared by Pictect-Spengler reactions, then Protectiveness coupling is carried out, obtains intermediate 4 and 5；In the 3- positions of B-carboline ring, different amino acid is introduced to produce a series of Yin Diindyl derivative 7a-s；Then, terminal acetylene functional group is introduced as synthesis " handle " to introduce TEMPO parts, passes through copper (I) it is catalyzed azide/alkynes cycloaddition reaction (CuAAC) (Le Droumaguet, C.； Wang,C.；Wang, Q.luorogenic clickreaction.Chem Soc Rev.39:1233-9；201037. Kawamoto,S.A.； Coleska,A.；Ran,X.；Yi,H.；Yang,C.Y.；et al.Design of triazole-stapled BCL9α- helical peptides to target theβ-catenin/B-cell CLL/lymphoma 9 (BCL9)protein- protein interaction.J.Med.Chem.55:1137-1146；2012.Yapici,N.B.； Mandalapu,S.R.； Chew,T.L.；Khuon,S.；Bi,L.Determination of intracellular pH using sensitive, clickable fluorescentprobes.Bioorg.Med.Chem.Lett.22:2440-2443；2012. Yapici, N.B.；Jockusch,S.；Moscatelli,A.；Mandalapu,S.R.；Itagaki,Y.；et al.New rhodamine nitroxide based fluorescentprobes for intracellular hydroxyl radical identification in livingcells.Org Lett.14:50-53；2012.) the object 9a-s needed for obtaining.

The various functional groups of these amino acid offer, including cation (Lys, Arg), aromatic group (Tyr, Trp, Phe), The different groups such as anion (Glu, Asp) and neutral fat group (Ser, Thr, Gly, Ala, Leu, Val, Ile).Use¹H With¹³C NMR verify to the structure of all intermediates.By HPLC, EPR (being confirmed the existence of nitrogen oxides part) and MS analyses To verify compound 9a-s purity, and the architectural feature of compound is demonstrated using high resolution mass spec method, the results showed that All target compounds (9a-s) are stable, and are stored at room temperature until using.

Synthetic route is as follows：

Wherein, reagent and condition are：I formaldehyde and sulfuric acid；IIBoc2O and triethylamine；III benzylalcohols, polyphosphoric acid, backflow；IV HOBt, DCC；VH₂, Pd/C；VI L-AA-OBzl, HOBt, DCC；VII HCl/EtOAc； VIII H₂, Pd/C；Propargylamine, HBTU, triethylamine；IX 4- azidos-TEMPO, CuI.

Further, the invention also provides described indoles-TEMPO conjugates weaken intestine ischemia/fill again preparing Note the purposes in the remote organ injury of induction.

To sum up, the present invention designs and has synthesized a series of new indoles-TEMPO conjugates (9a-s), and they have non-steroid Beneficial effect possessed by body anti-inflammatory agent (NSAID) and general antioxidant.The anti-inflammatory and antioxygen of the indoles-TEMPO conjugates Change effect can depend on two kinds of pharmacological properties：(i) anti-inflammatory power of indolyl moiety；(ii) TEMPO groups may have removing With inactivation mitochondria O₂ ^-·And other ROS ability.The study result show that indoles TEMPO conjugates (9f, i, j, P, r, s) as new pharmacophore there is sizable treatment potentiality, the I/R tissue damages induced can be down to most by they It is low.

In the present invention, we have studied give indoles-bis- TEMPO 9r, s in advance to mitigate the intestines of enteron aisle I/R inductions And including lung, the ability of the remote organ injury of liver and kidney.Although the mechanism of these compounds needs further research, It is that their protection activity may come from its anti-inflammatory, the anti-oxidant and ability of anti-apoptotic, this obtains indoles-TEMPO and is conjugated Thing can suppress 1) LPO generations；2) release of TNF-α；With these true supports of 3) mitochondrial cytochrome c release.So And, it is necessary to more study to study whether other cell factors (such as IL-1, IL-6) are participated in by intestine ischemia/Reperfusion injury Hinder induction multiple organ injury, and indoles-TEMPO conjugates whether can be protected during MOF it is another Individual vitals heart, it is allowed to not by intestines I/R damage influences.The answer of these problems can help us to more fully understand Yin The mechanism of diindyl TEMPO conjugates and optimize its use, individually or as a part for combined therapy agent to prevent or weaken intestines I/ MOF caused by R damages.

Brief description of the drawings

Fig. 1 is that dimethylbenzene induces dropsy of ear test method(s) to assess indoles-TEMPO conjugates (9a-s), Tempol (Tem) and The anti-inflammatory activity of its precursor (7a-s).Aspirin (Asp) is positive control.N=10；Oral aspirin dosage=20mg/ kg；Tempol, 7a-s and 9a-s oral dose are 0.10mmol/kg；*：Compared with aspirin, p<0.05；**：With Ah Take charge of a forest form ratio, p<0.01.

Fig. 2 evaluates the 9f, i, j, p, r of various dose (0.02,0.05,0.10mmol/kg), sization for dropsy of ear test method(s) The result of the anti-inflammatory activity of compoundN=10；*：Compound phase ratio identical with 0.02mmol/kg dosage, p< 0.05；**：Compound phase ratio identical with 0.02mmol/kg dosage, p<0.01.

Fig. 3 is indoles-TEMPO conjugates (9f, i, p, r, s) and its precursor 7f, i, j, p, r, s to active (the U/g groups of MPO Knit) influence.N=10.###：Compared with vehicle Control, p<0.001；***：Compared with dimethylbenzene+vehicle Control, p< 0.001；**：p<0.01；*：p<0.05.

Fig. 4 A are the redox reaction of indoles-TEMPO conjugates；Fig. 4 B indoles-TEMPO conjugates and its precursor The lowest energy conformation isomers and highest occupied molecular orbital (HOMO) energy partly illustrate in Fig. 4.

Fig. 5 is not have (C) or pre-processed with TEMPOL (Tem) or indoles-TEMPO conjugates (9f, i, j, p, r, s) Simulated ischemia-Reperfu- sion in cell death.Error bars represent the standard error of the average value of at least three independent experiments (compared with the control, * p<0.05).

Fig. 6 is the detection of the oxidative stress in mitochondria during simulated ischemia/Reperfu- sion (sI/R).(A) use The typical confocal fluorescence image for the HUVEC cells that MitoProbe, MitoTracker and Hoechst 33342 is dyed.In sI/R Period, mitochondria undergo different morphological changes；(B) by measuring the fluorescence intensity increase in different time points, line is determined O in plastochondria₂ ^-·Level.During sI/R, MitoProbe fluorescence is obvious relative to the increase of baseline value.With indoles-bis- TEMPO (9r, s) pre-processes the increase of MitoProbe fluorescence intensities during being obviously reduced sI/R.Error bars are represented from extremely Standard error (compared with the control, the * * p of the average value of few three independent experiments<0.01).

The release of cytochrome c during Fig. 7 is simulation I/R.(A) with MitoProbe by HUVEC cell dyeings.Simulating After ischemic or Reperfu- sion is after 3 hours, and cell is fixed on into baseline.Then immunostaining is carried out to cytochrome c；(B) will display The cell of cytochrome c redistribution is quantified and is expressed as the percentage of total cell colony.In experience simulation I/R cell In observe that the percentage for the cell that cytochrome c release occurs dramatically increases.Handled with indoles-bis- TEMPO (9r, s) Cell in cytochrome c release be obviously reduced.Error bars are represented from least standard error of the average value of independent experiment three times Difference (compared with the control, * p<0.05).

Fig. 8 is the representative microphoto of the small bowel tissue sections from I/R and I/R+9s treatment groups.(A)：H and E contaminates Color；(B)：Modified trichrome stain；(C) NADH is dyed.In I/R groups, the shortening of fine hair, the vacuolation of fluff tip, it will be apparent that The dissociation of lamina propria and the accumulation of polymorphonuclear leukocyte；With 9s processing mucosa injury is caused to be obviously reduced, only light spalling The fluff tip gland structure good with preservation.(D)：The caused influence (nmol/ μ g) of 9r, s on MDA； (E)：The influence of 9r, s to serum fingerprint (TNF-α)；In D, E, a：Compared with sham-operation, p<0.01；b：With Salt solution+I/R is compared, p<0.01.

Fig. 9 is the representative microphoto of the nephridial tissue section from I/R and I/R+9s groups.(A)：H and E is dyed；(B)： Modified trichrome stain；(C)：NADH is dyed.Representative histotomy is obtained from I/R groups mouse：Tubular necrosis, tubular structure Disintegration and Bowman capsules breaks are clearly visible in the tissue for being derived from I/R group mouse；Histotomy is derived from I/R+9s (30mg/ Kg) the mouse of processing：Most of tubules are complete, and in 9s treatment groups, renal tubular necrosis substantially reduces, and interstitial is expanded most It is few.D)：The influence of 9r, s to blood urea nitrogen (BUN) serum levels；(E)：The influence of 9r, s to creatinine (Cr) serum levels；D with E, a：Compared with sham-operation, p<0.01； b：Compared with salt solution+I/R, p<0.01.

Figure 10 is the representative microphoto of hepatic tissue section.(A)：H and E is dyed；(B)：Improve trichrome stain；(C) NADH is dyed.Representative histotomy is obtained from the mouse of I/R groups：It is obvious in the tissue taken out from I/R groups mouse to occur sternly The sinus property expansion of weight, bleeding, the central vein of thickening and the liver cell to degenerate；Obtained from the mouse of I/R+9s (30mg/kg) processing The histotomy taken：Expanded in the presence of slight sinus property；Central vein, vena portae hepatica and liver cell are acted normally in most of regions. D)：The influence of 9r, s to ALT (ALT) serum levels；(E)：9r, s are to aspartate aminotransferase (AST) influence of serum levels；In D and E, a：Compared with sham-operation, p<0.01；b：Compared with salt solution+I/R, p<0.01.

Figure 11 is the representative microphoto of lung tissue section.(A)：H and E is dyed；(B)：Improve trichrome stain；(C) NADH is dyed.The representative histotomy obtained from the mouse of ischemia/reperfusion：Bleeding is have recorded in I/R groups, significantly The disappearance of cellular infiltration and alveolar space；The mouse for I/R+9s (30mg/kg) treatments that histotomy obtains：Septal thickening and Neutrophil cell infiltration significantly improves after 9s interventions.

Figure 12 is the indoles-bis- TEMPOs and the mechanism of cytochrome c interaction proposed；

Figure 13 A-C are compound 7a-s and 9a-s molecular dynamics simulation result.

Embodiment

Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit Qualitatively, it is impossible to which protection scope of the present invention is limited with this.Unless otherwise indicated, all chemicals are generally purchased from Sigma Aldrich.All reactions are carried out under nitrogen atmosphere (1bar).Use TLC (60F₂₅₄The Merck silica gel plates of type, thickness 0.25mm) and HPLC (Waters, C₁₈Post 4.6mm × 150mm) confirm intermediate and final product purity (>97%).NMR Spectrum is obtained using the spectrometers of Bruker Advance 500.FAB-MS VG-ZAB-MS high-resolution GC/MS/DS and HP ES-5989x is determined.

The preparation of the 3S-1,2,3,4- tetrahydro-beta-carboline -3- carboxylic acids (1) of embodiment 1

25mL H are added in the solution of 5.0g (24.5mmol) L-Trp₂SO₄(1mol/L), 80mL water and 8mL first Aldehyde (36-38%).Reactant mixture is stirred at room temperature 2 hours, and adjusted with concentrated ammonia solution to pH 6-7.By gained Mixture is kept for 12 hours at 0 DEG C, and precipitation is collected by filtration.After recrystallization, 3.97g (75%) colorless powder is obtained Target compound.

Mp 280-282℃；EI/MS：217[M+H]⁺；IR(KBr)：3450,3200,3000,2950,2850,1700, 1601,1452,1070,900cm^-1；¹H NMR (BHSC-500, DMSO-d₆)：δ=10.99 (s, 1H), 9.89 (s, 1H), 7.30 (t, J=7.5Hz, 1H), 7.22 (t, J=8.0Hz, 1H), 7.01 (d, J=8.0Hz, 1H), 6.81 (d, J=7.5Hz, 1H), 4.01 (t, J=4.8Hz, 1H), 3.64 (dd, J=10.5Hz, J=2.4Hz, 1H), 2.91 (d, J=10.5Hz, 2H), 2.86 (s, 1H).

The preparation of embodiment 2N-Boc-3S-1,2,3,4- tetrahydro-beta-carboline -3- carboxylic acids (2)

1 suspension in 15mL DMF and 1.4mL triethylamines of 1.1g (5.0mmol) compound is violent at room temperature Stirring, then add 1.1g (7.7mmol) Boc-N₃And it is stirred for 30 minutes.It is small that reactant mixture is stirred at room temperature 24 When, then stirred 80 hours at 40 DEG C.5mL citric acid solutions (20%), solution ethyl acetate are added in reactant mixture (3 × 30mL) is extracted.The anhydrous MgSO of the ethyl acetate layer isolated₄Dry.MgSO is removed by filtration₄Afterwards, filtrate is led to Pervaporation is dried.Gained residue is in CHCl₃Middle crystallization, obtain 1.20g (76%) target compound.

Mp 165-170℃；¹H NMR (500MHz, DMSO-d₆)：δ=10.87 (s, 1H), 9.86 (s, 1H), 7.32 (t, J=7.6Hz, 1H), 7.21 (t, J=7.9Hz, 1H), 7.00 (d, J=7.9Hz, 1H), 6.84 (t, J=7.6Hz, 1H), 4.84 (t, J=5.0Hz, S-2 1H), 4.20 (dd, J=10.2Hz, J=4.8Hz, 1H), 3.98 (dd, J=10.2Hz, J= 3.2Hz, 1H), 2.93 (d, J=10.2Hz, 2H), 1.46 (s, 9H)

The preparation of embodiment 3 (S) -2,3,4,9- tetrahydrochysene -1H- pyrido [3,4-b] indole -3-carboxylic acid benzyl esters (3)

Under agitation, 0.02mol compounds 1 are added into the solution of 10.0g polyphosphoric acids and 50mL benzylalcohols.Will be anti- Answer mixture to be stirred 8 hours at 92 DEG C, then mixed in 200ml water with 20ml concentrated sulfuric acid solutions.Mixture 20ml second Ether processing, separates aqueous phase, is then neutralized with the aqueous sodium carbonate of regulation to pH10.It is molten that this is extracted with ether (3 × 70ml) Liquid, ether are mutually dried with anhydrous magnesium sulfate.After filtering, filtrate is settled out target compound 3, yield is with chlorination hydrogen blister 58%.

Mp：122-124℃；[α]_D ²⁵=53.4 (c=1.0, methanol)；ESI-MS(m/z)：307[M+H]⁺；¹H NMR (500MHz,DMSO-d₆)：δ/ppm=7.27-7.40 (m, 7H), 6.95 (t, J=7.5Hz, 1H), 7.02 (t, J=10Hz, 1H), 5.19 (s, 2H), 3.98 (q, J=15.5Hz, 2H), 3.82 (q, J=4.5Hz, J=8.5Hz, 1H), 2.97 (dd, J= 4.5Hz, J=15Hz, 1H), 2.82 (q, J=8.5Hz, J=15Hz).¹³C NMR(DMSO-d₆) δ/ppm=173.21, 136.57,136.23,133.46,128.90,128.71,128.46,128.21,127.30,120.97, 118.79, 117.73,111.33,105.83,66.15,55.63,41.70,25.26。

The 3- of embodiment 4 (3- ((benzyloxy) carbonyl) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -2- carbonyls Base) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-t-butyl formate (4) preparation

At 0 DEG C, 1.1mmol N- hydroxy benzos are added under stirring into compound 2 (1.0mmol) anhydrous THF solution Triazole (HOBT) and 1.1mmol N, N'- dicyclohexylcarbodiimides (DCC).After stirring 30 minutes, compound 3 is added The suspension of (1.1mmol) in the anhydrous THF of 5mL.Reactant mixture is adjusted to pH 8-9, Ran Hou with N-methylmorpholine It is stirred overnight at room temperature, until TLC shows that raw material has exhausted.After evaporation, residue is dissolved in 40mL ethyl acetate.Will be molten Liquid is fully washed with 5% sodium acid carbonate, 5%KHSO4 and saturated sodium-chloride.Separate organic layer and dried with anhydrous Na 2SO4.Slightly Residue it is filtered and be evaporated under reduced pressure after, by silica gel flash column chromatography, obtain compound 4, yield 80.6%.

Mp：230-232℃；[α]_D ²⁵=41.9 (c=1.0, methanol)；ESI-MS(m/z)：606[M+H]⁺；¹H NMR (500MHz,DMSO-d₆)：δ/ppm=10.82-10.97 (m, J=27.5Hz, J=49.5Hz, 2H), 6.93-7.52 (m, 13H),5.76(m,1H),5.61(m,1H),5.08-5.31(m,2H),4.49-5.08(m,4H), 3.41-3.57(m,2H), 2.95-3.18(m,2H),1.46(s,1H)。¹³C NMR(DMSO-d₆) δ/ppm=172.56,170.89,155.57, 136.92,130.92,129.65,128.67,127.63,126.91,121.74,119.24,118.90, 117.95, 111.40,80.58,66.75,65.41,55.25,52.17,41.71,28.48,24.58,22.03。

The 2- of embodiment 5 (2- (tertbutyloxycarbonyl) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) - The preparation of 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole -3-carboxylic acids (5)

Compound 4 (0.5mmol) is dissolved in 10mL MeOH and adds 40mg Pd/C, is passed through H2, is then stirred at room temperature, Until TLC shows that raw material has exhausted.Thick residue it is filtered and be evaporated under reduced pressure after, by silica gel flash column chromatography, obtain To compound 5, yield 83.5%.

Mp：245-248℃；[α]_D ²⁵=19.9 (c=1.0, methanol)；ESI-MS(m/z)：513[M-H]^-

The compound 6a-s of embodiment 6 preparation

Prepare 6a-s conventional method：Under 0 DEG C of stirring, to the anhydrous THF solution of compound 5 (514mg, 1.0mmol) Middle addition 1.2mmol N- hydroxybenzotriazoles (HOBT) and 1.2mmol N, N'- dicyclohexylcarbodiimides (DCC).Stirring After 10 minutes, suspension of the 1.2mmol amino-acid benzyls ester hydrochloride in the anhydrous THF of 5mL is added.By reactant mixture N- Methyl morpholine is adjusted to pH 8-9, is then stirred at room temperature overnight, until TLC shows that raw material has exhausted., will be residual after evaporation Excess is dissolved in 40mL ethyl acetate.By solution 5% sodium acid carbonate, 5%KHSO₄Fully washed with saturated sodium-chloride.Separation Organic layer simultaneously uses anhydrous Na₂SO₄Dry.Thick residue is filtered and after being evaporated under reduced pressure, by silica gel flash column chromatography, Obtain compound 6a-s.

(1) 3- ((S) -3- ((2- (benzyloxy) -2- oxoethyls) carbamoyl) -2,3,4,9- tetrahydrochysene -1H- pyridines And [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-formic acid-(S)-tert-butyl ester (6a)：

Yield：43%.Mp：145-148℃；[α]_D ²⁵=12.6 (c=1.0, methanol)；ESI-MS(m/z)： 663[M+H ]⁺。¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.84-10.91 (m, 2H)；8.56(m,1H), 7.18-7.43(m, 9H),6.93-7.09(m,4H),5.18-5.69(m,2H),4.61-5.18(m,5H),4.45(m, 1H),3.65-4.11(m, 2H),2.81-3.51(m,4H),1.46(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=171.95,170.97,169.84, 157.20,155.22,136.84,131.13,129.37,128.82,127.05,126.98,121.40 ,118.98, 118.03,111.59,105.49,80.63,66.30,56.32,51.24, 41.36,38.87,33.96,28.35,22.99, 20.08。

(2) 3- ((S) -3- (((R) -1- (benzyloxy) -1- oxo propyl- 2- yls) carbamoyl) -2,3,4,9- tetrahydrochysenes - 1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic acid-(S)-uncle Butyl ester (6b)：

Yield：51%.Mp：146-148℃；[α]_D ²⁵=-29.4 (c=1.0, methanol)；ESI-MS(m/z)676[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.81-10.88 (m, 2H)；8.42(m,1H),7.18-7.43(m, 9H),6.89-7.09(m,4H),5.64(m,1H),5.35(m,1H),5.07(m,1H),4.81-4.99(m,3H), 4.45- 4.79(m,3H),4.26(m,1H),3.12-3.45(m,3H),2.89-3.10(m,1H),1.47(s,9H), 1.30-1.37 (m,3H)。¹³C NMR(DMSO-d₆) δ/ppm=172.55,171.96,170.96,170.16,155.32,136.77, 131.56,128.74,128.40,128.09,126.91,121.23,118.88,117.98,111.40,105.02, 80.54, 66.19,54.24,50.67,48.46,48.04,41.49,28.47,24.94,24.01,17.40。

(3) 3- ((S) -3- (((R) -1- (benzyloxy) -3- methyl isophthalic acids-oxo butyl -2) carbamoyl) -2,3,4,9- Tetrahydrochysene (4,4- dihydro -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H) carboxylic acid-(S)-tert-butyl ester (6c)：

Yield：55%.Mp：125-127℃；[α]_D ²⁵=-18.0 (c=1.0, methanol)；ESI-MS(m/z)704[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.81-10.88 (m, 2H)；8.22(m,1H),7.18-7.43(m, 9H),6.93-7.08(m,4H),5.65(m,1H),5.36(m,1H),4.31-5.29(m,6H),4.15(m,1H), 2.81- 3.50(m,5H),1.47(s,9H),0.83-0.92(m,6H)。¹³C NMR(DMSO-d₆) δ/ppm=172.64,172.07, 170.93,156.05,155.25,136.82,130.59,129.26,128.79,127.19,126.97,121.21 , 118.87,117.96,111.57,105.25,80.61,66.58,54.60,52.49, 52.26,41.73,35.51,28.44, 23.71,22.99,14.40。

(4) 3- ((S) -3- (((2R, 3R) -1- (benzyloxy) -3- methyl isophthalic acids-oxopentyl -2) carbamoyl) -2,3, 4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic Acid-(S)-tert-butyl ester (6d)：

Yield：48%.Mp：133.7-135℃；[α]_D ²⁵=-23.0 (c=1.0, methanol)；ESI-MS(m/z)： 718[M +H]⁺。¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.82-10.91 (m, 2H), 8.29 (m, 1H), 7.18-7.43 (m, 9H),7.79-7.80(m,4H),5.68(m,1H),5.42(m,1H),4.98-5.21(m,2H), 4.51-4.98(m,4H), 4.25(m,1H),2.93-3.18(m,5H),1.47(s,9H),1.19-1.31(m,2H), 0.81-0.86(m,3H),0.63- 0.67(m,3H)。¹³C NMR(DMSO-d₆) δ/ppm=172.51,171.44,170.68,155.87,155.38,136.79, 130.71,128.77,128.52,128.42,126.92,121.32,121.21, 118.86,117.94,111.38,80.46, 68.98,66.35,66.15,53.71,36.86,36.58,33.82,28.46, 25.81,24.48,23.95,15.82。

(5) 3- ((S) -3- (((R) -1- (the benzyloxy) -4- methyl isophthalic acids-amyl- 2- yls of oxo) carbamoyl) -2,3,4, 9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic Acid-(S)-tert-butyl ester (6e)：

Yield：55%.Mp：138-140℃；[α]_D ²⁵=-26.1 (c=1.0, methanol)；ESI-MS(m/z)718[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.81-10.88 (m, 2H)；8.32(m,1H),7.15-7.44(m, 9H),6.93-7.09(m,4H),5.64(m,1H),5.35(m,1H),4.39-5.15(m,6H),4.21(m,1H), 2.85- 3.49(m,4H),1.51-1.79(m,3H),1.49(s,9H),0.63-0.88(m,6H)。¹³C NMR(DMSO-d₆) δ/ppm= 173.00,172.42,171.51,156.00,155.33,136.76,130.52,128.77, 128.47,128.25, 126.91,121.21,118.86,117.97,111.54,105.18,80.54,66.36,65.40, 54.06,50.93, 41.91,41.39,28.45,25.36,24.54,23.70,22.21。

(6) 3- ((S) -3- (((R) -4- amino -1- (benzyloxy) -1,4- dioxo butane -2- bases) carbamoyl) - 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic acid (S) tert-butyl ester (6f)：

Yield：61%.Mp：162-164℃；[α]_D ²⁵=-2.5 (c=1.0, methanol)；ESI-MS(m/z)719[M+H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.82-10.90 (m, 2H)；8.32(m,1H),7.18-7.50(m, 9H), 6.89-7.10(m,4H),5.65(m,1H),5.44(m,1H),4.41-5.23(m,7H),2.90-3.50(m, 6H),1.47 (s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=206.95,172.34,171.60,171.28,170.47,155.33, 136.75,131.15,129.94,128.70,127.96,126.92,121.21,118.87,117.94,111.37,105.32, 80.55,66.27,56.51,53.75,49.37,41.54,39.15,36.75,28.33,23.70,23.01。

(7) 3- ((S) -3- (((R) -1- (benzyloxy) -3- (1H- imidazol-4 yls) -1- oxo propyl- 2- yls) carbamyls Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles - 2 (9H)-carboxylic acids-(S)-tert-butyl ester (6g)：

Yield：64%.Mp：180-182℃；[α]_D ²⁵=-8.0 (c=1.0, methanol)；ESI-MS(m/z)742[M+H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.84-10.82 (m, J=5Hz, 2H)；8.58 (m, J=7Hz, 1H), 7.96 (s, 1H), 7.18-7.42 (m, 9H), 6.89-7.11 (m, 5H), 5.65 (m, J=1.5Hz, 1H), 5.34 (m, J=16.6Hz, 1H),4.68-5.12(m,5H),4.31-4.52(m,2H),2.89-3.48(m,6H),1.48(s,9H)。 ¹³C NMR(DMSO- d₆) δ/ppm=172.24,171.06,170.60,163.42,155.26,136.77,135.01,132 .52,131.16, 129.91,128.73,128.32,126.91,125.99,121.21,118.87,117.95,116.88,114.41,105.37, 80.59,66.33,65.36,54.53,52.91,51.24,41.43,32.78,28.47,23.68,22.86。

(8) 3- ((S) -3- (((R) -1- (benzyloxy) -1- oxo -3- phenyl propyl- 2- yls) carbamoyl) -2,3,4, 9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic acid The tert-butyl ester (6h):

Yield：58%.Mp：133-135℃；[α]_D ²⁵=-23.0 (c=1.0, methanol)；ESI-MS(m/z)752[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.76-10.90 (m, 2H)；8.35(m,1H),7.18-7.42(m, 14H),6.89-7.11(m,4H),5.63(m,1H),5.35(m,1H),4.68-5.19(m,5H),4.21-4.65(m, 1H), 2.85-3.49(m,6H),1.41(m,9H)。¹³C NMR(DMSO-d₆) δ/ppm=171.45,171.29,170.85, 155.27,137.59,136.51,136.09,131.01,129.97,129.46,128.71,128.28,126.89,121.21 ,118.87,117.97,111.39,104.30,80.53,66.32,54.42,51.01,37.03,36.58,28.49,23.58, 22.99。

(9) 3- ((R) -1- (benzyloxy) -6- (((benzyloxy) carbonyl) amino) -1- oxo hex- 2- yls) carbamyl Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles - 2 (9H)-carboxylic acids (the S)-tert-butyl ester (6i)：

Yield：57%.Mp：116-118℃；[α]_D ²⁵=-20.4 (c=1.0, methanol)；ESI-MS(m/z)867[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.82-10.89 (m, J=10Hz, 2H)；8.25-8.45(m, 2H), 7.18-7.43(m,14H),6.9-07.14(m,4H),5.64(m,1H),5.34(m,1H),4.51-5.19(m, 8H),4.29 (m,1H),2.89-3.55(m,6H),1.56-2.11(m,4H),1.46(s,9H),1.09-1.11(m, 2H)。¹³C NMR (DMSO-d₆) δ/ppm=172.84,172.03,171.23,169.90,156.52,155.97,141.64,133 .79, 130.12,128.98,128.78,128.16,126.92,121.21,118.88,117.94,111.39,105.11,80.54, 67.61,66.31,65.58,57.81,54.06,53.51,52.98,50.77,32.17,30.76,28.48,25.43, 23.77,21.95。

(10) 3- ((S) -3- (((R) -1- (benzyloxy) -3- (4- hydroxy phenyls) -1- oxo propyl- 2- yls) carbamyls Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles - 2 (9H)-carboxylic acids (the S)-tert-butyl ester (6j)：

Yield：66%.Mp：157-159℃；[α]_D ²⁵=-17.2 (c=1.0, methanol)；ESI-MS(m/z)768[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.80-10.91 (m, 2H)；9.33(s,1H),8.55(m,1H), 7.18-7.42(m,9H),6.45-7.10(m,8H),5.64(m,1H),5.38(m,1H),4.66-5.15(m,6H), 4.47 (m,1H),3.55-3.98(m,2H),2.89-3.45(m,4H),1.46(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm= 171.56,171.44,170.93,156.50,155.29,136.78,136.53,131.18, 130.43,130.02, 129.62,129.35,128.69,128.39,127.33,122.33,121.222,120.66,118.89, 117.95, 111.58,104.33,80.53,66.34,66.20,54.94,50.71,42.02,38.86,36.42,28.54, 23.65, 22.55。

(11) 3- ((S) -3- (((R) -1- (benzyloxy) -4- (methyl mercapto) -1- oxo-butanes -2- bases) carbamyls Base) -2,3,4,9- (3,4- dihydro -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] Indoles -2 (9H)-carboxylic acid-(the S)-tert-butyl ester (6k)：

Yield：65%.Mp：131-133℃；[α]_D ²⁵=-16.5 (c=1.0, methanol)； ESI-MS(m/z)736[M+H ]⁺。¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.80-10.88 (m, J=24.5Hz, 2H)；8.23 (m, J=8.0Hz, 1H), 7.16-7.47 (m, 9H), 6.89-7.08 (m, 4H), 5.23-5.83 (m, J=6.5Hz, 1H), 4.91-5.13 (m, J= 12Hz, J=12.5Hz, 3H), 4.63-4.9 (m, 2H), 4.27-4.62 (m, 2H), 2.89-3.61 (m, 4H), 1.98-2.49 (m,4H),1.61-1.94(m,3H),1.42(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=172.69,171.87, 170.82,156.44,155.94,136.81,130.14,129.36,128.85,128.48,126.93,121.36 , 118.88,117.97,111.58,105.32,80.80,66.49,56.33,55.01,54.10,51.68,41.89,30.97, 29.96, 28.50,23.84,22.94,14.94。

(12) 3- ((S) -3- ((double (the benzyloxy) -1- oxo propyl- 2- yls of (R) -1,3-) carbamoyl) -2,3,4,9- Tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic acid - (S)-tert-butyl ester (61)：

Yield：67%.Mp：120-122℃；[α]_D ²⁵=-15.4 (c=1.0, methanol)；ESI-MS(m/z)782[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.82-10.91 (m, J=16Hz, 2H)；8.52 (m, J=8.0 Hz, 1H), 7.15-7.43 (m, 14H), 6.97-7.09 (m, 4H), 5.68 (m, J=6.5Hz, 1H), 5.37 (m, J=4.5 Hz, 1H), 4.26-5.09 (m, 9H), 3.51-3.90 (m, 4.5Hz, 2H), 2.95-3.45 (m, J=6.5Hz, J=15.5Hz, 4H),1.45(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=173.02,172.38,171.16,170.74,155.75, 138.22,136.79,130.78,129.17,128.72,128.64,128.17,128.09,127.96,126.93, 121.35,118.90,117.94,111.59,105.01,80.46,72.84,69.79,69.47,66.56,66.42,53.74, 41.92,41.92,28.49,24.56,22.31。

(13) 3- ((S) -3- (((R) -1- (benzyloxy) -3- (1H- indol-3-yls) -1- oxo propyl- 2- yls) carbamyls Base) -2,3,4,9- (3,4- dihydro -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] Indoles -2 (9H)-carboxylic acid-(the S)-tert-butyl ester (6m)：

Yield：54%.Mp：155-156℃；[α]_D ²⁵=-17.8 (c=1.0, methanol)；ESI-MS(m/z)791[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.78-10.90 (m, J=24.5Hz, 3H)；8.56(m,1H), 7.19-7.52(m,9H),6.90-7.09(m,9H),5.64(m,1H),5.36(m,1H),4.51-5.20(m,6H), 4.32 (m,1H),2.90-3.45(m,6H),1.41(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=171.86,171.06, 170.01,155.27,136.53,136.07,30.40,128.66,128.27,128.06,127.51,126.90, 124.17, 121.50,118.91,118.42,111.59,105.20,80.53,66.32, 60.21,56.33,54.16,50.93, 42.01,38.86,28.57,23.73,22.44。

(14) 3- ((S) -3- ((R) -2- ((benzyloxy) carbonyl) pyrrolidines -1- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridines And [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H)-carboxylic acid (S)-tert-butyl ester (6n)：

Yield：61%.Mp：150-152℃；[α]_D ²⁵=-2.7 (c=1.0, methanol)；ESI-MS(m/z)702[M+H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.73-10.92 (m, J=27.5Hz, 2H)；7.48 (m, J=8Hz, 3H), 7.41 (m, J=7.5Hz, 1H), 7.19-7.37 (m, 7H), 6.98-7.09 (m, 4H), 5.64 (m, 1H), 5.32 (m, 1H), 5.11 (t, J=17Hz, 1H), 4.86-4.89 (m, 2H), 4.74-4.81 (m, 2H), 4.66 (m, 1H), 4.18 (dd, J= 7Hz,1H),2.96-3.35(m,4H),2.74-2.89(m,2H),1.452.18(m,4H),1.44(s, 9H)。¹³C NMR (DMSO-d₆) δ/ppm=172.11,171.89,168.97,157.13,155.61,136.66,131.51,129 .69, 128.78,128.06,126.99,121.27,118.92,117.81,111.58,105.90,80.41, 66.06,59.34, 52.04,50.90,49.83,48.01,46.65,41.96,33.82,28.52,24.93,23.00,21.65。

(15) (S) -3- ((S) -3- ((double (the benzyloxy) -1- oxo-butanes -2- bases of (2R, 3R) -1,3-) carbamyls Base) -2,3,4,9 tetrahydrochysenes (3,4- dihydro -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3, 4-b] indoles -2 (9H)-carboxylic acid-(the S)-tert-butyl ester (6o):

Yield：55%.Mp：123-125℃；[α]_D ²⁵=-19.1 (c=1.0, methanol)；ESI-MS(m/z)796[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.82-10.91 (m, 2H)；8.3 (m, J=9Hz, 1H), 7.18- 7.41(m,14H),6.89-7.17(m,4H),5.68(m,1H),5.47(m,1H),4.49-5.21(m,7H), 3.96-4.49 (m,3H),2.89-3.41(m,4H),1.46(s,9H),0.83-1.28(m,3H)。¹³C NMR(DMSO-d₆) δ/ppm= 173.22,172.59,171.98,170.46,155.59,138.57,136.52,131.06, 130.06,128.73, 128.59,128.45,128.05,126.93,121.21,118.87,117.86,111.38,105.07, 80.50,74.45, 70.68,66.52,56.79,53.15,50.51,42.00,41.54,28.48,25.16,22.27,16.13。

(16) 3- ((S) -3- ((the amyl- 2- yls of (R) -1- (benzyloxy) -5- (3- nitros guanidine radicals) -1- oxos) carbamyls Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles - 2 (9H)-carboxylic acids-(S)-tert-butyl ester (6p)：

Yield：68%.Mp：170-172℃；[α]_D ²⁵=-22.1 (c=1.0, methanol)；ESI-MS(m/z)806[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.84-10.89 (m, 2H)；8.56(m,1H),7.10-7.44(m, 9H),6.89-7.10(m,4H),5.63(m,1H),5.37(m,1H),4.69-5.20(m,6H),4.49(m,1H), 2.89- 3.51(m,6H),1.51-1.89(s,4H),1.47(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=171.85,171.27, 159.80,155.53,136.78,136.50,130.13,129.43,128.77,126.91,122.30,121.23, 118.89,117.94,112.93,105.05,80.56,67.40,66.40,54.75,52.48,50.74,31.78,31.14, 29.45,28.47,25.34,23.83,22.24。

(17) 3- ((S) -3- ((the amyl- 2- yls of (R) -5- amino -1- (benzyloxy) -1,5- dioxos) carbamoyl) -2, 3,4,9 tetrahydrochysenes-(1H- pyridos [3,4-b] indole-2-carbonyl) -3,4- dihydro -1H- pyridos [3,4-b] indoles -2 (9H) - Carboxylic acid-(S)-tert-butyl ester (6q)：

Yield：57%.Mp：158-159℃；[α]_D ²⁵=-12.1 (c=1.0, methanol)；ESI-MS(m/z)733[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.83-10.91 (m, 2H)；8.44 (m, J=7.5Hz, 1H), 7.19- 7.46 (m, 9H), 6.89-7.10 (m, 4H), 5.65 (m, J=7Hz, 1H), 5.31 (m, 1H), 4.61-5.12 (m, 5H), 4.41 (m,1H),4.23(m,1H),3.11-3.51(m,3H),2.99(m,1H),1.99-2.21(m,2H), 1.78-1.95(m, 2H),1.47(s,9H)。¹³C NMR(DMSO-d₆) δ/ppm=173.90,172.77,171.88,171.17,170.61, 155.95,136.75,130.65,128.95,128.85,128.34,128.12,126.90,121.26, 118.90, 117.96,111.57,105.17,80.70,66.43,53.94,52.53,52.22,50.67,41.75,31.63, 28.55, 27.23,26.59,23.88。

(18) 2- ((S) -2- ((S) -2- tert-butoxycarbonyl -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b]-carbonyl) - 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups)-(R)-Dibenzyl succinate (6r)：

Yield：59%.Mp：114-117℃；[α]_D ²⁵=-10.9 (c=1.0, methanol)；ESI-MS(m/z)810[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.83-10.88 (m, J=4Hz, 2H)；8.54 (m, J=8Hz, 1H), 7.18-7.43(m,14H),6.89-7.15(m,4H),5.64(m,1H),5.34(m,1H),4.64-5.21(m, 8H),4.41 (m, J=6Hz, 1H), 2.52-3.45 (m, 6H), 1.47 (s, 9H).¹³C NMR(DMSO-d₆) δ/ppm=172.21, 171.62,170.68,165.24,155.90,155.28,136.83,130.05,129.31,128.82,128.16, 126.94,121.35,118.88,117.95,111.58,105.24,80.64,69.12,66.85,66.38,56.33, 51.07, 48.81,41.44,36.31,28.96,23.32,22.37。

(19) 2- ((S) -2- ((S) -2- tert-butoxycarbonyls -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] carbonyl) - 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) -2- (R)-glutaric acid dibenzyl ester (6s)：

Yield：62%.Mp：114-116℃；[α]_D ²⁵=-15.2 (c=1.0, methanol)；ESI-MS(m/z)824[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=10.81-10.88 (m, 2H)；8.39 (m, J=7.5Hz, 1H), 7.18- 7.43(m,14H),6.89-7.11(m,4H),5.67(m,1H),5.25(m,1H),4.31-5.29(m,6H), 4.15(m, 1H),2.81-3.50(m,5H),1.47(s,9H),0.83-0.92(m,6H)。¹³C NMR(DMSO-d₆) δ/ppm=172.64, 172.07,170.93,156.05,155.25,136.82,130.59,129.26, 128.79,127.19,126.97, 121.21,118.87,117.96,111.57,105.25,80.61,66.58,54.60, 52.49,52.26,41.73, 35.51,28.44,23.71,22.99,14.40。

The compound 7a-s of embodiment 7 preparation

Prepare compound 7a-s conventional method：1mL is added in 1.0mmol 6a-s ethyl acetate (5mL) solution HCl/EtOAc.Reactant mixture is stirred at room temperature, until TLC shows that raw material has exhausted.After evaporation, residue passes through silicon Glue flash column chromatography, obtain compound 7a-s.

(1) 2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- four Hydrogen -1H- pyridos [3,4-b] indoles -3- formamidos) benzyl acetate (7a)：

Yield：89%.Mp：180-183℃；[α]_D ²⁵=-74.4 (c=1.0, methanol)；ESI-MS(m/z)562[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.29 (m, 1H), 10.25 (m, 1H), 9.85 (m, 1H), 8.75 (m, 1H), 7.25-7.56 (m, 9H), 6.89-7.18 (m, 4H), 5.35 (m, J=5.9Hz, 2H), 5.10-5.25 (m, 2H), 4.98-5.04 (m, 2H), 4.35-4.55 (m, 2H), 3.75-4.05 (m, J=5.6Hz, 3H), 3.31-3.50 (m, J= 14.7Hz, J=4.3Hz, 2H), 2.89-3.18 (m, J=6.3Hz, 2H).¹³C NMR(DMSO-d₆) δ/ppm=172.35, 170.02,169.63,136.81,136.22,129.84,128.94,128.36,127.09,126.30,122.18, 119.63,118.03,111.85,105.21,67.29,66.39,55.10,48.00,33.80,23.26,21.51.

(2) 2- (S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- four Hydrogen -1H- pyridos [3,4-b] indoles -3- formamido groups) propionic acid-(R)-benzyl ester (7b):

Yield：87%.Mp：176-178℃；[α]_D ²⁵=-77.6 (c=1.0, methanol)；ESI-MS(m/z)576[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.34 (m, 1H), 10.35 (m, 1H), 9.88 (m, 1H), 8.68 (m, 1H), 7.15-7.56 (m, 9H), 6.98-7.10 (m, 4H), 5.25-5.49 (m, J=7.5Hz, 2H), 5.05-5.20 (m, 2H), 4.81-4.92 (m, 2H), 4.25-4.52 (m, J=8.5Hz, 3H), 3.25-3.49 (m, J=3.5Hz, 2H), 2.89- 3.10 (m, J=15.5Hz, J=7.5Hz, 2H), 1.46-1.48 (m, 3H).¹³C NMR(DMSO-d₆) δ/ppm=172.41, 170.71,169.60,136.78,136.31,130.05,128.97,128.50,128.07,126.22,122.19, 121.37,119.60,111.96,105.18,67.43,66.26,54.52,48.59,33.81,24.91,21.55,17.43.

(3) 3- methyl -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -1, 2,3,4- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) butyric acid-(R)-benzyl ester (7c)：

Yield：90%.Mp：168-170℃；[α]_D ²⁵=-98.9 (c=1.0, methanol)；ESI-MS(m/z)604[M+H ]⁺； 1H NMR(500MHz,DMSO-d₆)：δ/ppm=11.37 (m, 1H), 10.25 (m, 1H), 9.89 (m, 1H), 8.56 (m, 1H), 7.18-7.59 (m, 9H), 6.98-7.15 (m, 4H), 5.10-5.35 (m, J=10Hz, 3H), 4.75-4.96 (m, J= 10Hz, 3H), 4.35-4.65 (m, J=15Hz, 3H), 3.35-3.51 (m, 3H), 2.89-3.15 (m, J=15Hz, 2H), 0.81-0.89(m,6H)。¹³C NMR(DMSO-d₆) δ/ppm=172.35,171.46,169.69,136.81,136.22, 129.55,128.95,128.79,128.48,127.17,122.19,121.47,119.59,111.95, 104.94,67.52, 66.35,58.47,57.78,50.86,30.08,24.32,21.53,17.98。

(4) 2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- four Hydrogen -1H- pyridos [3,4-b] indoles -3- formamido groups)-(2R, 3R) -3- methvl-pentanoic acids benzyl ester (7d)：

Yield：87%.Mp：166-168℃；[α]_D ²⁵=-129.0 (c=1.0, methanol)； ESI-MS(m/z)618[M+ H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.37 (m, 1H), 10.35 (m, 1H), 9.85 (m, 1H), 8.65 (m, 1H), 7.18-7.56 (m, 9H), 6.98-7.15 (m, 4H), 5.05-5.45 (m, J=7.05Hz, 3H), 4.75-4.95 (m, J =12.3Hz, J=10.5Hz, 2H), 4.35-4.65 (m, 3H), 4.21 (m, J=6.45Hz, 1H), 3.28-3.48 (m, J= 4.15Hz, J=4.75Hz, 3H), 2.89-3.15 (m, 2H), 1.31-1.55 (m, 2H), 0.79-0.89 (m, 6H).¹³C NMR (DMSO-d₆) δ/ppm=172.34,171.27,169.68,136.82,136.20,129.53,128.93,128 .77, 127.17,126.24,122.19,119.60,118.27,111.96,105.14, 67.50,57.06,53.93,50.88, 42.30,36.87,25.84,24.98,21.52,15.89,11.57。

(5) 4- methyl -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2, 3,4,5- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) valeric acid-(R)-benzyl ester (7e)：

Yield：92%.Mp：156-158℃；[α]_D ²⁵=-57.3 (c=1.0, methanol)；ESI-MS(m/z)618[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.36 (m, 1H), 10.30 (m, 1H)；9.85(m,1H),8.65 (m, 1H), 7.21-7.57 (m, 9H), 6.98-7.20 (m, J=5Hz, J=10Hz, 4H), 5.35 (m, J=10Hz, 1H), 5.05- 5.28(m,2H),4.78-4.95(m,3H),4.25-4.55(m,3H),3.30-3.49(m,2H),2.89-3.25 (m,2H), 1.55-1.79(m,2H),1.50(m,1H),0.77-0.95(m,6H)。¹³C NMR(DMSO-d₆) δ/ppm=172.42, 172.39,169.63,136.78,136.30,129.49,128.97,128.69, 127.16,122.19,119.60, 117.92,111.97,104.97,67.52,66.29,54.60,50.98,42.22,40.88, 33.79,24.90,23.19, 22.44,21.56。

(6) 4- amino -4- oxos -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] carbonyls of indoles -3 Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamidos) butyric acid-(R)-benzyl ester (7f)：

Yield：92%.Mp：194-196℃；[α]_D ²⁵=-137.0 (c=1.0, methanol)； ESI-MS(m/z)619[M+ H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.28 (m, 1H), 10.95 (m, 1H), 10.15 (m, 1H), 10.25 (m, 1H), 9.85 (m, 1H), 8.55 (m, J=13.5Hz, 1H), 8.45 (m, 1H), 7.15-7.57 (m, 9H), 6.98-7.14 (m, 4H), 5.05-5.45 (m, J=6.5Hz, 2H), 4.89-5.03 (m, J=3Hz, J=12Hz, 2H), 4.81 (m, 1H), 4.35-4.69 (m, J=10Hz, 4H), 2.89-3.51 (m, 4H), 2.55-2.65 (m, 2H).¹³C NMR(DMSO-d₆)δ/ppm =172.38,171.38,170.10,169.46,136.79,136.32,130.02,129.23,128.75,127.04, 126.25,122.20,121.37,119.03,111.87,105.13, 66.42,54.62,51.18,49.68,41.97, 37.05,36.76,23.24,21.52。

(7) 3- (1H- imidazol-4 yls) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles - 3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) propionic acid-(R)-benzyl ester (7g)：

Yield：93%.Mp：183-185℃；[α]_D ²⁵=-72.5 (c=1.0, methanol)；ESI-MS(m/z)642[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.25 (m, 1H), 10.98 (m, 1H), 10.05 (m, 1H), 9.25 (m, 1H), 8.98 (m, 1H), 7.55 (dd, J=5Hz, 1H), 7.20-7.49 (m, 9H), 6.98-7.18 (m, 4H), 5.25-5.45 (m, J=5Hz, 2H), 5.01-5.23 (m, 2H), 4.98 (m, J=15Hz, 3H), 4.89 (m, J=15Hz, 2H), 4.35- 4.68(m,3H),3.28-3.48(m,2H),2.89-3.25(m,4H)。¹³C NMR(DMSO-d₆) δ/ppm=171.40, 170.18,169.42,136.82,136.06,135.43,135.22,130.67, 129.41,128.82,126.82, 122.19,121.39,120.04,119.53,111.96,105.17,66.74,65.88, 54.94,51.27,42.11, 38.98,25.97,23.57,22.37。

(8) 3- phenyl -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2, 3,4,9 tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) propionic acid-(R)-benzyl ester (7h)：

Yield：82%.Mp：154-156℃；[α]_D ²⁵=-88.3 (c=1.0, methanol)；ESI-MS(m/z)652[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.35 (m, 1H), 10.89 (m, 1H), 9.98 (m, 1H), 8.85 (m, 1H),7.15-7.57(m,14H),6.98-7.12(m,4H),5.12-5.45(m,2H),4.90-5.10(m,3H), 4.75(m, 1H), 3.30-3.51 (m, 2H), 2.89-3.29 (m, J=3.5Hz, 2H), 2.89-3.10 (m, J=15.5Hz, J=7.5Hz, 2H),1.46-1.48(m,3H)。¹³C NMR(DMSO-d₆) δ/ppm=172.36,170.55,169.30,137.77, 136.84,136.18,129.89,129.67,129.44,129.04,128.81,128.67,128.32,128.11, 127.68,122.22,119.41,118.01,111.55,105.05。

(9) 6- (((benzyloxy) carbonyl) amino) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Indoles -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) caproic acid-(R)-benzyl ester (7i)：

Yield：85%.Mp：134-136℃；[α]_D ²⁵=-54.0 (c=1.0, methanol)；ESI-MS(m/z)767[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.28 (m, 1H), 10.95 (m, 1H), 10.20 (m, 1H), 9.85 (m, 1H), 8.57 (m, 1H), 7.21-7.56 (m, 14H), 6.98-7.20 (m, 4H), 5.15-5.35 (m, J=10.5Hz, 2H), 4.95-5.10 (m, J=6.5Hz, 4H), 4.75-4.90 (m, J=14Hz, 2H), 4.05-4.55 (m, J=9.5Hz, J= 12Hz, 3H), 3.15-3.51 (m3H), 2.89-3.12 (m, J=10.5Hz, J=15.5Hz, 3H), 1.65-1.78 (m, 2H), 1.21-1.49(m,4H)。¹³C NMR(DMSO-d₆) δ/ppm=172.35,171.84,169.82,156.55,136.81, 136.29,130.01,129.49,128.77,128.16,127.09,122.22,119.61,118.04,111.62,105.33, 67.53,66.26,65.58,54.60,53.03,52.35,50.93,42.23,30.88,29.42,23.03,21.51。

(10) 3- (4- hydroxy phenyls) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b]-carbonyl) - 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamidos) propionic acid-(R)-benzyl ester (7j)：

Yield：83%.Mp：175-178℃；[α]_D ²⁵=-77.6 (c=1.0, methanol)；ESI-MS(m/z)668[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.25 (m, 1H), 10.90 (m, 1H), 10.25 (m, 1H), 9.95 (m, 1H), 8.65 (m, 1H), 7.21-7.56 (m, 9H), 6.89-7.20 (m, 6H), 6.58-6.75 (m, J=5Hz, 2H), 5.12- 5.35 (m, 2H), 5.05 (m, J=15Hz, 1H), 4.65-4.98 (m, 3H), 4.25-4.48 (m, 3H), 3.25-3.45 (m, 2H),2.89-3.20(m,4H)。¹³C NMR(DMSO-d₆) δ/ppm=172.43,170.63,169.49,156.54, 136.79,136.14,13.88,130.45,129.38,128.78,128.24,128.18,127.60, 122.27,121.54, 119.07,115.64,111.66,105.23,67.51,55.12,54.78,42.01,36.05,35.68, 23.60,21.51。

(11) 4- (methyl mercapto) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] -2,3,4,9- four Hydrogen -1H- pyridos [3,4-b] indoles -3- formamidos) butyric acid-(R)-benzyl ester (7k)：

Yield：88%.Mp：165-168℃；[α]_D ²⁵=-82.5 (c=1.0, methanol)；ESI-MS(m/z)635[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.27 (m, 1H), 10.30 (m, 1H), 9.85 (m, 1H), 8.65 (m, 1H),7.21-7.56(m,9H),6.88-7.20(m,4H),4.95-5.45(m,6H),4.35-4.52(m,3H), 3.25- 3.45(m,2H),2.89-3.15(m,2H),2.352.45(m,2H),2.05-2.15(m,2H),1.85-1.95(m, 3H)。¹³C NMR(DMSO-d₆) δ/ppm=172.35,170.72,169.54,136.81,136.27,129.92,129.57,128 .87, 128.20,127.10,126.23,122.22,121.39,118.63,111.63,105.24,66.52, 56.49,54.57, 52.07,42.28,31.05,30.13,28.82,23.51,21.51,14.89。

(12) 3- (benzyloxy) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) propionic acid-(R)-benzyl ester (7l)：

Yield：87%.Mp：148-150℃；[α]_D ²⁵=-78.8 (c=1.0, methanol)；ESI-MS(m/z)682[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.30 (m, 1H), 10.20 (m, 1H), 9.85 (m, 1H), 8.78 (m, 1H), 7.15-7.56 (m, 9H), 6.98-7.12 (m, 4H), 5.01-5.35 (m, J=11Hz, J=9.5Hz, 2H), 4.75- 5.00 (m, J=11.5Hz, 2H), 4.25-4.65 (m, 6H), 3.15-3.58 (m, 4H), 2.89-3.10 (m, 2H).¹³C NMR (DMSO-d₆) δ/ppm=172.41,170.15,169.60,137.78,136.77,136.15,129.47,128 .90, 128.13,127.98,127.13,126.76,126.21,122.22,121.48,119.02,111.62,105.21, 72.96, 69.61,66.55,54.54,51.31,42.22,24.71,21.53。

(13) 3- (1H- indol-3-yls) -2- (2- (2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) - 2,3,4,9 tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) benzyl propionate (7m)：

Yield：89%.Mp：184-186℃；[α]_D ²⁵=-67.4 (c=1.0, methanol)；ESI-MS(m/z)691[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.36 (m, 1H), 10.97 (m, 1H), 10.25 (m, 1H), 9.89 (m, 1H),8.75(m,1H),7.15-7.57(m,13H),6.98-7.13(m,5H),4.98-5.35(m,2H), 4.87(m,1H), 4.71(m,1H),4.25-4.55(m,4H),4.10(m,1H),3.31-3.49(m,2H), 3.15-3.29(m,2H),2.89- 3.14(m,2H)。¹³C NMR(DMSO-d₆) δ/ppm=172.43,171.87,169.78,136.78,136.62,136.14, 129.97,129.52,128.74,128.07,127.11,124.26,122.20, 121.66,119.59,111.95, 109.83,105.07,67.51,66.30,54.60,53.25,48.01,33.80,24.91, 21.55。

(14) 1- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9 four Hydrogen -1H- pyridos [3,4-b] indoles -3- carbonyls) pyrrolidines -2- carboxylic acids-(R)-benzyl ester (7n)：

Yield：85%.Mp：169-171℃；[α]_D ²⁵=-62.4 (c=1.0, methanol)；ESI-MS(m/z)602[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.32 (m, 1H), 10.25 (m, 1H), 9.85 (m, 1H), 7.15- 7.57(m,9H),6.98-7.13(m,4H),5.11-5.35(m,2H),4.55-5.09(m,2H),4.25-4.55 (m,5H), 3.61(m,1H),3.15-3.49(m,4H),3.01(m,1H),1.72(m,1H),1.63(m,1H), 1.15-1.25(m,2H) 。¹³C NMR(DMSO-d₆) δ/ppm=172.41,171.50,169.10,136.73,136.29,130.05,128.96, 128.40,128.15,126.88,122.17,119.51,118.36,111.85,104.55,67.73, 66.45,59.05, 58.95,47.97,45.68,33.79,28.20,24.89,22.41,21.54。

(15) (3R) -3- (benzyloxy) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles - 3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) butyric acid-(2R)-benzyl ester (7o)：

Yield：85%.Mp：154-156℃；[α]_D ²⁵=-64.7 (c=1.0, methanol)；ESI-MS(m/z)696[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.37 (m, 1H), 10.25 (m, 1H), 9.89 (m, 1H), 8.45 (m, 1H), 7.19-7.56 (m, 14H), 6.89-7.15 (m, 4H), 5.05-5.48 (m, 3H), 4.98 (m, J=3Hz, 2H), 4.89 (m, 1H), 4.51-4.75 (m, 2H), 4.254.45 (m, 5H), 3.25-3.45 (m, J=4Hz, J=2Hz, 2H), 2.89- 3.10 (m, J=12Hz, 2H), 1.14-1.19 (m, 3H)¹³C NMR(DMSO-d₆) δ/ppm=172.42,170.41, 169.75,138.48,136.78,136.00,129.42,128.91,128.63,128.59,128.49,128.03, 127.87,126.72,122.20,121.49,119.04,111.95,105.18,74.73,72.86,67.67,66.78, 57.05, 54.58,50.78,41.02,39.86,22.23,21.55,16.51.

(16) (R)-(3- nitros guanidine radicals) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b]-carbonyl Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) benzyl valerianate (7p)：

Yield：87%.Mp：174-175℃；[α]_D ²⁵=-26.7 (c=1.0, methanol)；ESI-MS(m/z)706[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.27 (m, 1H), 10.96 (m, 1H), 9.89 (m, 1H), 8.65 (m, 1H),7.89-8.15(m,2H),7.13-7.56(m,9H),6.98-7.10(m,4H),4.78-5.45(m,9H), 4.15- 4.55(m,2H),2.89-3.45(m,4H),1.65-1.85(m,2H),1.45-1.55(m,2H)。¹³C NMR(DMSO-d₆)δ/ Ppm=172.38,171.70,169.61,159.80,136.80,136.22,130.00,128 .78,128.18,127.06, 126.23,122.22,121.58,119.04,111.62,105.06,66.36,54.61,52.73, 50.99,42.24, 28.41,25.50,24.07,23.62,21.51。

(17) 5- amino-5-oxos -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyrido [3,4-b] indoles -3- Carbonyl) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) valeric acid-(R)-benzyl ester (7q)：

Yield：81%.Mp：179-181℃；[α]_D ²⁵=-80.3 (c=1.0, methanol)；ESI-MS(m/z)633[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.30 (m, 1H), 10.95 (m, 1H), 10.56 (m, 1H), 10.25 (m,1H),9.85(m,1H),8.75(m,1H),7.21-7.56(m,9H),6.98-7.20(m,4H), 5.05-5.35(m, 4H),4.75-4.95(m,2H),4.15-4.55(m,3H),3.15-3.52(m,2H),2.89-3.12 (m,2H),2.15- 2.25(m,4H)。¹³C NMR(DMSO-d₆) δ/ppm=174.13,172.44,171.70,169.62,136.76,136.27, 130.06,129.47,128.53,128.17,127.10,122.19,119.51,118.04,111.55, 105.06,66.32, 56.99,54.55,51.01,32.14,31.89,31.86,28.72,25.11,21.55。

(18) (R) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3, 4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) Dibenzyl succinate (7r)：

Yield：80%.Mp：140-142℃；[α]_D ²⁵=-103.7 (c=1.0, methanol)； ESI-MS(m/z)710[M+ H]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.33 (m, 1H), 10.25 (m, 1H), 9.89 (m, 1H), 8.85 (m, 1H),7.22-7.56(m,14H),6.96-7.20(m,4H),5.35(m, 1H),5.20(m,1H),5.15(m,1H),4.65- 5.05(m,5H),4.35-4.49(m,3H),3.35-3.50(m, 2H),2.80-3.25(m,4H)。¹³C NMR(DMSO-d₆)δ/ Ppm=172.36,170.52,170.29,169.46,136.83,136.06,128.90,128 .79,128.52,128.38, 126.92,122.21,119.43,117.96,111.57, 105.25,67.82,66.88,54.79,49.44,41.09, 40.88,39.36,36.16,23.49,21.52。

(19) (R) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3, 4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamido groups) glutaric acid dibenzyl ester (7s)：

Yield：82%.Mp：135-137℃；[α]_D ²⁵=-96.4 (c=1.0, methanol)；ESI-MS(m/z)724[M+H ]⁺；¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.27 (m, 1H), 10.25 (m, 1H), 9.89 (m, 1H), 8.69 (m, 1H), 7.19-7.56 (m, 14H), 6.98-7.18 (m, 4H), 5.30 (m, J=6.5Hz, 1H), 5.21 (m, J=3.5Hz, 1H), 4.95-5.15 (m, 4H), 4.85-4.95 (m, 2H), 4.21-4.55 (m, 3H), 3.15-3.45 (m, J=6.5Hz, 2H), 2.89-3.12 (m, J=12Hz, 2H), 2.35-2.49 (m, 2H), 1.95-2.18 (m, 2H).¹³C NMR(DMSO-d₆)δ/ Ppm=173.21,172.59,171.38,169.80,136.77,136.23,129.94,129 .45,128.78,128.31, 128.17,127.08,122.22,121.39,119.03,111.87,105.21,67.66,66.44, 65.97,52.28, 50.98,30.49,26.46,24.10,21.53。

The compound 8a-s of embodiment 8 preparation

Compound 8a-s's is typically prepared method：Added in 1.0mmol 7a-s absolute ethyl alcohol (140mL) solution 0.20g Pd/C.In H₂Reactant mixture is stirred at room temperature in atmosphere, until TLC instruction parent materials exhaust.Will reaction Mixture is filtered by celite posts, uses CH₂Cl₂Washing, it is evaporated in vacuo the filtrate of merging.Residue is dissolved in CH₂Cl₂In, Washed twice with saturated brine solution.By organic layer anhydrous Na₂SO₄Dry, filter and concentrate.

Residue is dissolved in the anhydrous CH of 10mL₂Cl₂In, and 2- (1H- BTA -1- bases) 1,1,3,3- is supplemented successively Tetramethylurea hexafluorophosphate (HBTU) 0.5g (1.4mmol), propargylamine 0.09mL (1.4mmol) and triethylamine 0.25mL (1.4mmol).Reactant mixture is stirred at room temperature overnight, until TLC instruction parent materials exhaust.By reactant mixture Use CH₂Cl₂Dilution, then uses salt water washing.Merge organic layer, with anhydrous sodium sulfate drying, filter and be evaporated under reduced pressure.By remnants Thing is directly used in next reactions steps without purifying in addition.

4- azidos-Tempo：In ice bath by 1.1g NaN₃(17.0mmol) is dissolved in 5mL water, adds toluene 5mL.With Trifluoromethanesulfanhydride anhydride 1.7mL (10.4mmol) is added dropwise afterwards, reaction stirred is added dropwise until the completion of TLC Indicator Reactions NaHCO₃Solution is until bubbling stops.Reactant mixture is extracted with toluene (10mL × 2), organic layer anhydrous sodium sulfate drying. Toluene is not evaporated, because it is reported that when no solvent is present, trifluoromethyl azide has explosivity.By 1.08g 4- ammonia Base-Tempo (5.85mmol) is dissolved in 7.5mL water, then adds 2.0g NaHCO₃(23.8mmol).Then, 100mg is added CuSO₄(0.63mmol), then add 12mL fluoroform sulfuryl azide stock solutions.Reactant is stirred at room temperature 3 Hour, use NaHCO₃Solution is washed and extracted with EtOAc.Organic layer is separated, uses anhydrous sodium sulfate drying.After evaporation, residue Pass through fast silica gel chromatogram post EtOAc:Hexane (1:3) purifying is rinsed, obtains 1g orange solids shape target compound (80% yield).¹H NMR(400MHz,CDCl₃)δppm：1.15 (s, 6H), 1.21 (s, 6H), 1.52 (t, J=12.2Hz, 2H), 1.85 (dd, J=11.8,2.9Hz, 2H), 3.58 (tt, J=12.2,3.8Hz, 1H).¹³C NMR(100MHz, CDCl₃)δppm20.1,32.5,44.3,52.9,59.1。

The compound 9a-s of embodiment 9 preparation

Prepare compound 9a-s conventional method：At room temperature in 0.5mmol compound 8a-s and 0.6mmol 4- nitrine 15.2mg cupric iodides (I) (0.08mmol) are added in solution of the base-Tempo in 3mL acetonitriles.By reactant mixture in room temperature Lower stirring 6 hours.Reactant mixture saturation NaHCO₃The aqueous solution (3 × 10mL) wash, then with ethyl acetate (30mL × 3) extract.By the organic layer anhydrous sodium sulfate drying of merging, filter and concentrate.Thick residue is passed through into the quick post color of silica gel Spectrometry purifies, and obtains target compound 9a-s.

(1) (S)-N- (2- (((1- (1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles -4- Base) methyl) amino) -2- oxoethyls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) - 2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9a)：

Yield：57%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.52 (brs), 10.84 (brs), 9.87 (brs), 8.85(brs),7.60(brs),7.21-7.45(brs),6.90-7.20 4.98-5.04(brs),4.20-4.56(brs), 3.75-3.95(brs),2.0(brs),1.20(brs).It was observed that paramagnetism it is broadening be due to NO free radical portion in molecule The influence divided.¹³C NMR(100Hz,DMSO-d₆) δ/ppm=172.3,170.0,169.1,136.8,136.3,130.5, 129.4,128.8,127.6,123.1,126.5,122.6,119.7,121.2,119.0,118.2,117.3,111.8, 105.1, 72.1,68.3,56.7,50.7,37.8,33.4,43.5,36.7,37.8,23.4,22.9。HR/MS[M+H]⁺Calculate Value 706.3625, measured value 706.3697.

(2) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -1- oxo propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls Base) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9b)：

Yield：68%.¹H NMR(400MHz,CDCl₃),δ/ppm 11.46(brs),10.87(brs),8.97(brs), 8.62(brs),7.62(brs),6.91-7.20(brs),7.21-7.45(brs),4.95(brs),4.45-4.65(brs), 3.75-3.95(brs),2.93-3.10(brs),2.0(brs),1.49(brs),1.21(brs).It was observed that paramagnetism it is broadening It is due to the influence of NO free radical part in molecule.¹³C NMR(DMSO-d₆) δ/ppm=172.5,170.5,169.3, 130.4,131.5,136.2,137.2,130.5,123.0,127.6,126.7,122.0,121.5,120.3,121.8, 112.1, 110.1,119.0,105.1,72.0,69.0,51.5,38.4,31.8,56.4,55.5,23.1,25.7,37.6, 36.6,44.5, 25.4,23.2,18.2.Implement HR/MS [M+H]⁺Calculated value 720.3782, measured value 720.3851.

(3) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -3- methyl isophthalic acids-oxo-butanes -2- bases) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Indoles -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9c)：

Yield：55%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.55 (brs), 10.87 (brs), 9.07 (brs), 8.78(brs),7.63(brs),7.22-7.40(brs),6.90-7.20 4.95-5.02(brs),4.45-4.55(brs), 3.70-3.95(brs),2.95-3.03(brs),2.0(brs),1.21(brs),1.01(brs).It was observed that paramagnetism it is broadening It is due to the influence of NO free radical part in molecule.¹³C NMR(DMSO-d₆) δ/ppm=170.5,171.3,169.8, 131.2,135.8,134.3,131.5,129.7,128.5,123.2,126.4,127.4,122.5,122.6,120.2, 120.3, 112.3,110.9,119.0,117.5,105.2,105.7,72.3,69.0,63.2,56.3,54.7,50.7, 45.1,38.2, 33.5,37.5,36.8,35.8,25.3,32.2,23.3,18.7。HR/MS[M+H]⁺Calculated value 748.4095, Measured value 748.4157.

(4) (S)-N- ((2R, 3R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- three Azoles -4- bases) methyl) amino) -3- methyl isophthalic acids-oxo-pentane -2- bases) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3, 4-b] -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9d)：

Yield：47%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.50 (brs), 10.98 (brs), 8.97 (brs), 8.65(brs),7.62(brs),6.92-7.20(brs),7.21-7.40 4.95-5.04(brs),4.35-4.53(brs), 3.75-3.95(brs),2.94-3.10(brs),2.53(brs),2.0(brs),1.60(brs),1.15-1.20(brs), 1.05(brs).It was observed that paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=172.2,170.7,169.8,131.7,132.5,137.5,135.3,131.4,123. 1,128.4,127.5, 122.3,122.1,120.3,119.7,112.6,110.1,119.0,118.7,105.0,104.7,72.6, 70.1,64.1, 54.2,56.7,51.4,44.1,38.7,33.7,37.8,36.9,23.8,37.9,26.3,25.2,23.6,24.5, 15.2, 11.6。HR/MS[M+H]⁺Calculated value 762.4251 [M+H]⁺, measured value：762.4312.

(5) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -4- methyl isophthalic acids-oxo-pentane -2- bases) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Indoles -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9e)：

Yield：51%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.50 (brs), 10.88 (brs), 9.17 (brs), 8.67(brs),7.62(brs),6.92-7.20(brs),7.21-7.35(brs),4.95(brs),4.45-4.55(brs), 3.70-3.90(brs),2.95-3.05(brs),2.0(brs),1.52-1.75(brs),1.20(brs),1.01(brs).See The paramagnetism observed is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm= 171.8, 170.5,169.5,131.7,130.2,137.5,135.7,131.4,128.7,123.7,126.9,123.0, 122.6,121.4, 120.3,112.1,110.9,117.8,118.0,105.7,104.2,72.1,69.0,58.4,56.7, 55.8,52.1,50.5, 45.1,41.7,38.4,37.3,36.5,33.5,23.7,26.2,25.5,23.2,24.5,23.3, 22.5。HR/MS[M+H]⁺Calculated value 762.4251, measured value：762.4297.

(6)(R)-N¹- ((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazole-4-yls) first Base) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysenes -1H- Pyrido [3,4-b] indoles -3- formamidos) succinamide (9f)：

Yield：56%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.53 (brs), 10.83 (brs), 9.08 (brs), 8.67(brs),7.64(brs),7.05-7.23(brs),7.02(brs),6.95-7.20(brs),7.21-7.40(brs), 4.85-4.95(brs),4.45-4.55(brs),3.70-3.90(brs),2.93-3.05(brs),2.77(brs),2.01 1.20(brs).It was observed that paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=172.7,172.2,170.6,169.6,138.1,135.0,132.7,130.5,131. 3,124.0,128.1, 126.3,122.5,121.3,120.5,120.9,119.4,111.7,112.1,105.1,104.1,119.0, 73.2,70.1, 52.0,56.7,54.2,55.6,50.5,45.7,38.1,33.5,37.8,37.6,23.4,23.7,22.4,24.3, 26.5。 HR/MS[M+H]⁺Calculated value 763.3840.Measured value：763.3877.

(7) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -3- (1H- imidazol-4 yls) -1- oxo propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridines And [3,4-b] carbonyl) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9g)：

Yield：47%.¹H NMR(400MHz,CDCl₃), δ/ppm=11.50 (brs), 10.85 (brs), 8.97 (brs), 8.52(brs),7.60-7.65(brs),7.21-7.45(brs),4.92-4.96(brs),4.45-4.55(brs),3.70- 3.95(brs), 2.95-3.10(brs),2.0(br),1.20(brs).It was observed that paramagnetism it is broadening be due to nitrogen in molecule The influence of oxygen radical part.¹³C NMR(DMSO-d₆) δ/ppm=171.7,170.5,169.3,137.6,136.3, 135.8,132.1, 131.6,132.1,132.8,123.6,128.0,122.4,123.1,120.1,121.4,112.4, 112.5,120.8,119.0, 118.6,106.1,105.5,72.0,69.0,57.6,52.1,38.3,33.4,57.2,56.3, 58.3,38.3,37.5,45.5, 32.3,30.5,24.3,25.3,23.3。HR/MS[M+H]⁺Calculated value 786.4000.Actual measurement Value：786.4076.

(8) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -1- oxo -3- phenyl propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Yin Diindyl -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9h)：

Yield：52%.¹HNMR(400MHz,CDCl₃), δ/ppm=11.50 (brs), 10.87 (brs), 8.95 (brs), 8.67(brs),7.62(brs),6.92-7.20(brs),7.21-7.45(brs),4.95-5.05(brs),4.45-4.57 (brs), 2.95-3.25(brs),3.70-3.90(brs),2.00(brs),1.20(brs).It was observed that paramagnetism broadening be Due to the influence of NO free radical part in molecule.¹³C NMR(DMSO-d₆) δ/ppm=172.5,171.0,169.7, 136.5,137.0, 136.4,131.6,132.8,131.3,123.4,128.5,126.9,120.7,122.3,123.5, 121.5,129.5,129.0, 127.4,128.8,128.0,119.6,112.4,113.5,119.3,106.3,105.2, 72.0,69.0,51.6,59.0,56.2, 57.5,37.3,35.8,44.5,38.2,38.4,33.6,25.5,24.2,24.5, 23.4,22.5。HR/MS[M+H]⁺Calculated value 796.4095.Measured value：796.4136.

(9) (S)-N- ((R) -6- amino -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2, 3- triazole -4- bases) methyl) amino) -1- oxo hex- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Yin Diindyl -3- carbonyls) -2,3,4,9-1,2,3,4- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9i)：

Yield：58%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.37 (brs), 10.97 (brs), 8.98 (brs),8.55(brs),7.65(brs),7.05-7.21(brs),4.45-4.65(brs),4.75-4.90(brs), 3.75- 3.95(brs),2.70-2.96(brs),2.0(brs),1.55-1.80(brs),1.21-1.26(brs).It was observed that paramagnetic Property is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=171.8,170.5, 169.3,131.5,130.8,135.8,137.4,132.0,123.5,128.1,126.9,122.3,122.8,120.4, 119.4, 112.4,110.9,119.3,118.2,105.1,106.3,72.6,69.5,51.4,38.4,33.2,55.6, 38.7,33.4, 57.4,56.8,23.4,26.5,37.4,36.4,44.5,58.6,43.2,32.7,29.2,23.6,22.6。 HR/MS[M+H]⁺Calculated value 777.4360, measured value 777.4425.

(10) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -3- (4- hydroxy phenyls) -1- oxo propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- (carbonyl) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9j)：

Yield：41%.¹H NMR(400MHz,DMSO-d6):D/ppm=11.55 (brs), 10.98 (brs), 8.97 (brs), 8.55(brs),6.92-7.20(brs),7.21-7.45(brs),7.65(brs),6.80-7.15(brs),4.95 (brs), 4.45-4.65(brs),3.70-3.90(brs),3.20-3.45(brs),2.90-3.10(brs),2.0(brs), 1.20(brs).It was observed that paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=172.4,170.6,169.7,156.7,136.4,131.2,132.4,130.2,129. 4,127.3,122.8, 121.17,121.7,111.5,118.8,116.0,119.8,104.9,71.9,69.2,56.7,56.5, 50.6,37.6, 32.0,43.1,37.4,36.6,25.1,23.2,22.2。HR/MS[M+H]⁺Calculated value 812.4044.Measured value 812.4092.

(11) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -4- (methyl mercapto) -1- oxo-butanes -2- bases) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3, 4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9k)：

Yield：49%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.52 (brs), 10.88 (brs), 8.97 (brs),8.65(m,1H),7.65(brs),6.91-7.20(brs),7.21-7.45(brs),4.95-5.05(brs), 4.45-4.55(brs),3.70-3.95(brs),2.90-3.10(brs),2.05-2.60(brs),2.00-2.10(brs), 1.20(brs).It was observed that paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=170.3,169.7,172.4,136.8,136.3,130.2,130.6,130.5,123. 7,128.3,127.1, 121.7,122.8,120.8,120.3,119.7,118.4,112.6,110.5,105.3,104.9,72.0, 69.3,58.6, 56.0,50.5,37.6,25.1,37.2,32.0,43.1,32.4,25.5,23.3,22.7,15.6。 HR/MS[M+H]⁺Calculate Value 780.3815.Measured value, 780.3887.

(12) (S)-N- ((R) -3- hydroxyls -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2, 3- triazole-4-yls) methyl) amino) -1- oxo propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] Yin Diindyl -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9l)：

Yield：51%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.55 (brs), 10.88 (brs), 8.95 (brs),8.72(brs),7.63(brs),6.92-7.20brs),7.21-7.45(brs),4.95(brs),4.20-4.56 (brs), 3.70-3.92(brs),2.90-3.10(brs),2.00(brs),1.2(brs).It was observed that paramagnetism broadening be Due to the influence of NO free radical part in molecule.¹³C NMR(DMSO-d₆) δ/ppm=172.8,170.2,167.4, 130.4,135.5, 136.7,130.5,127.8,127.3,128.1,125.1,121.9,102.4,119.0,111.6, 102.3,105.0,70.7, 66.3,61.7,60.5,50.5,56.5,44.2,35.4,25.5,23.3,22.0。HR/MS[M+ H]⁺Calculated value 736.3731, measured value 736.3798.

(13) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) -3- (1H- indol-3-yls) -1- oxo propyl- 2- yls) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridines And [3,4-b] pyridine -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9m)：

Yield：43%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.57 (brs), 11.02 (brs), 10.82 (brs),8.98(brs),8.75(brs),7.21-7.60brs),6.92-7.20(brs),4.98(brs),4.25-4.55 (brs), 3.70-3.95(brs),3.31-3.40(brs),2.90-3.10(brs),1.95(brs),1.20(brs).Observation To paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=172.4, 171.7, 169.2,136.7,135.9,130.5,135.6,130.8,128.1,124.1,123.7,127.9,126.5, 105.3,102.1, 127.4,109.4,49.5,35.2,30.3,55.7,50.4,70.5,67.6,111.9,111.0, 119.2,120.3,120.5, 20.5,23.2,119.7,121.7,120.8,36.7,40.5,57.8,28.7,25.9,23.3, 20.9。HS/MS[M+H]⁺835.4204 measured value 835.4279.

(14) (R)-N- ((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazole-4-yls) first Base) amino) -1- (S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- four Hydrogen -1H- pyridos [3,4-b] indoles -3- carbonyls) pyrrolidines -2- formamides (9n)：

Yield：38%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.52 (brs), 10.25 (brs), 8.90 (brs),7.65(brs),7.45(brs),6.98-7.20(brs),4.95(brs),4.25-4.55(brs),3.65-3.95 (brs), 2.95-3.10(brs),2.00-2.35(brs),1.20(brs).It was observed that paramagnetism it is broadening be due in molecule The influence of NO free radical part.¹³C NMR(DMSO-d₆) δ/ppm=170.8,169.2,167.8,136.2,135.5, 131.5, 130.6,135.7,130.8,123.5,124.2,128.2,122.6,127.6,122.8,128.9,120.4, 111.0,111.3, 120.5,119.2,105.2,102.2,108.9,70.3,69.2,60.9,50.5,48.5,43.0, 35.3,30.5,20.7,23.5, 37.2,24.6,22.4。HR/MS[M+H]⁺Calculated value 746.3938, measured value 746.4007.

(15) (S)-N- ((2R, 3R) -3- hydroxyls -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1, 2,3- triazole-4-yls) methyl) amino) -1- oxo-butanes -2- bases) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4- B] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9o)：

Yield：51%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.57 (brs), 10.65 (brs), 8.92 (brs),8.57(brs),6.92-7.20(brs),7.21-7.45brs),7.65(brs),4.95(brs),4.45-4.65 (brs), 3.75-3.95(brs),2.90-3.10(brs),1.95(brs),1.1-1.2(brs).It was observed that paramagnetism increase Width is due to the influence of NO free radical part in molecule.¹³C NMR(DMSO-d₆) δ/ppm=172.4,171.5,169.7, 131.5, 129.7,135.7,136.6,130.5,124.5,126.7,121.6,120.7,120.5,119.8,111.0, 111.5,105.4, 105.7,72.8,67.8,65.8,63.2,60.9,55.6,51.5,43.5,38.3,36.7,30.9, 22.7,36.8,33.0, 41.3,23.3。19.5。HR/MS[M+H]⁺Calculated value 750.3887, measured value 750.3943.

(16) (S)-N- ((R) -1- (((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles - 4- yls) methyl) amino) the amyl- 2- yls of -5- (3- nitros guanidine radicals) -1- oxos) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- formamides (9p)：

Yield：47%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.54 (brs), 11.02 (brs), 8.97 (brs),8.65(brs),7.63(brs),6.90-7.20brs),7.21-7.45(brs),4.98(brs),4.45-4.55 (brs), 3.75-3.95(brs),2.95-3.35(brs),2.0(brs),1.55-1.80(brs),1.20(brs).It was observed that Paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm=172.4, 171.70, 169.61,159.8,136.8,136.2,130.0,131.3,128.8,128.2,127.2,126.7,122.6, 121.6,120.6, 111.6,110.7,105.1,72.0,69.4,55.6,51.5,38.6,36.9,50.7,43.6,40.5, 29.1,25.6,24.1, 22.9,23.2。HR/MS[M+H]⁺Calculated value 850.4273, measured value 850.4345.

(17)(R)-N¹- ((1- (1- epoxide -2,2,6,6- tetramethyl piperazine -4- bases) -1H-1,2,3- triazole-4-yls) first Base) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysenes -1H- Pyrido [3,4-b] indoles -3- formamides) glutaramide (9q)：

Yield：54%.¹H NMR(500MHz,DMSO-d₆)：δ/ppm=11.50 (brs), 10.95 (brs), 8.95 (brs),8.75(brs),7.20(brs),7.21-7.56(brs),7.65(brs),4.45-4.65(brs),3.70-3.95 (brs), 2.80-3.12(brs),1.93-2.10(brs),1.2(brs).It was observed that paramagnetism it is broadening be due in molecule The influence of NO free radical part.¹³C NMR(DMSO-d₆) δ/ppm=174.1,172.4,171.7,169.6,136.7, 136.2, 130.6,129.5,127.5,128.3,127.1,123.4,122.2,119.6,118.4,111.5,111.2, 105.2,104.9, 72.0,69.3,56.9,55.5,50.5,36.7,32.6,28.7,25.1,23.3。HR/MS[M+H]⁺Meter Calculation value 777.3996, measured value 777.4065.

(18)N¹,N⁴- bis- ((1- (1- epoxide -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazole-4-yls) first Base) -2- ((S) -2- ((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysenes -1H- Pyrido [3,4-b] indoles -3- formamidos) succinamide (9r)：

Yield：45%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.60 (brs), 10.89 (brs), 8.98 (brs),8.65(brs),8.42(brs),7.60-7.65brs),6.97-7.20(brs),7.21-7.45(brs), 4.15- 4.55(brs),4.55-4.96(brs),3.70-3.95(brs),2.75-3.10(brs),1.93-2.0(brs),1.20 (brs)。 ¹³C NMR(DMSO-d₆) δ/ppm=173.8,172.5,170.3,169.8,136.8,136.2,131.2,131.0, 130.9,128.4,128.0,123.6,122.5,121.8,120.3,118.7,111.0,111.5,105.6,72.4,70.7, 56.5,55.0,50.7,43.5,44.0,36.6,39.0,37.8,32.9,25.6,23.3,22.5。HR/MS[M+H]⁺Calculate Value 998.5399, measured value, 998.5450.

(19)(R)-N¹,N⁵- bis- ((1- (1- oxo -2,2,6,6- tetramethyl piperidine -4- bases) -1H-1,2,3- triazoles -4- Base) methyl) -2- ((S)-((S) -2,3,4,9- tetrahydrochysene -1H- pyridos [3,4-b] indoles -3- carbonyls) -2,3,4,9- tetrahydrochysenes - 1H- pyridos [3,4-b] indoles -3- formamido groups) glutaramide (9s)：

Yield：49%.¹H NMR(400MHz,DMSO-d₆)：δ/ppm=11.57 (brs), 11.02 (brs), 9.02 (brs),8.90(brs),8.50(brs),7.60-7.65(brs),6.90-7.22(brs),7.23-7.46(brs),4.97 (brs), 4.20-4.58(brs),3.70-3.95(brs),2.93-3.15(brs),1.95-2.10(brs),1.2(brs)。 It was observed that paramagnetism it is broadening be due to NO free radical part in molecule influence.¹³C NMR(DMSO-d₆) δ/ppm= 174.1,171.3, 170.4,169.8,136.7,136.2,129.2,130.7,131.4,127.5,128.3,128.2, 127.3,123.2,121.7, 119.03,111.9,105.2,72.0,69.7,57.2,56.4,50.5,43.5,37.6, 36.7,33.5,28.0,23.3。 HR/MS[M+H]⁺Calculated value 1012.5555, measured value 1012.5632.

The purposes that the indoles-TEMPO conjugates 9a-s of embodiment 10 is far apart in organ confrontation ischemical reperfusion injury in protection

1st, method

1.1 cell culture

Human umbilical vein endothelial cells (HUVEC) added with endothelial cell growth replenishers, 5% hyclone and penicillin/ In the Endothelial cell culture base of Streptomycin Solution, at 37 DEG C, 5%CO₂Incubator in cultivate.

1.2 simulated ischemias/Reperfu- sion (sI/R) scheme

SI/R is realized in circulation chamber.In short, cell is washed with phosphate buffer solution (PBS), and it is molten in balance salt Liquid (116mmol/L NaCl, 5.4mmol/L KCl, 0.8mmol/L MgSO₄, 1mmol/L NaH₂PO₄, 0.9mmol/L CaCl₂It is phenol red with 10mg/L) in be incubated.Then by cell in the anoxic room equipped with Compact gas Control for Oxygen Content device Incubated in (Themo scientific, USA), with by injecting 94%N₂And 5%CO₂Admixture of gas 2 hours keep Oxygen concentration is 1%.After anoxic culture, cell is transferred back in oxygen containing complete medium.Normal control cells are in normal oxygen condition Under incubated in regular growth incubator.Before simulated ischemia, by cell and contain indoles-bis- TEMPOs9r, s (30 μ g/mL) Full culture medium incubated 12 hours under the conditions of normal oxygen.Discard the complete medium containing medicine.Cell is rinsed with PBS once, And the balanced salt solution containing indoles-bis- TEMPO9r, s (30mg/mL) is used to incubate 2 hours during simulated ischemia.Discard balance Salting liquid, then cell is incubated 3 hours under the conditions of normal oxygen in the complete medium of not drug containing.

1.3 molecular dynamics simulation

Molecular dynamics evaluation (CambridgeSoft Chem and Bio 3D 12.0) is carried out based on the MM2 field of forces.Use Some important parameters below：Step interval：2.0fs；Frame period：10fs；Terminated after 10000 steps；Heat/cool rates： 1.000Kcal/atom/ps；Target temperature：300°K.

1.4 biologicall test

" the Guide to the Care and Use of that all zooperies are announced according to NIH Laboratory Animals " are carried out.

1.5 interior anti-inflammatory activities are evaluated

The anti-inflammatory activity of compound synthesized by the dropsy of ear test method(s) evaluation of dimethylbenzene induction.In short, by male ICR mouse (20 ± 2g) are randomly divided into three groups, including test group, vehicle Control (1% cmc soln, CMC) and sun Property control group (aspirin).Positive controls receive oral suspension of the aspirin in CMC (10mg/kg).Test group Initial oral suspension (0.10mmol/kg) of the acceptance test material in CMC.After 30 minutes, 0.03ml dimethylbenzene is applied The forward and backward surface of auris dextra is added to, left ear is used as control.Two hours after dimethylbenzene is applied, mouse is put to death, takes out two ears. Multiple circular cross-sections are obtained using cork borer and are weighed.By subtracting untreated tissue from cutting in block weight for processing Block weight is cut to determine the weight increase related to dimethylbenzene stimulation.Myeloperoxidase (MPO) activity

Determine related myeloperoxidase (MPO) activity of tissue, be it is a kind of be used for measuring neutrophil cell infiltration and The indirect estimation methods of oxidative damage, carried out according to a kind of method being previously reported by.As a result U/g tissues are expressed as.

1.6 tail bleeding times measured

Indoles-the TEMPO newly synthesized is orally administered to male mice (body weight 18-22g) with 0.50mmol/kg dosage Conjugate (9a-s).After administration 30,45,60 and 90 minutes, mouse is placed in pipe holder, stretches out its afterbody, in tail Portion cuts out 2mm otch.The every 30 seconds blood that flowing is gently wiped with paper handkerchief is until stopped bleeding and records the time.

1.7 intestine ischemias/re-perfusion model

Male Wistar mouse (250 ± 50g) is randomly divided into five experimental groups：(1) sham-operation group：With except ischemic/ The mouse (n=8) of the following programs processing of Reperfu- sion (I/R) outside；(2) I/R groups：It is subjected to intestine ischemia 45 minutes, then Reperfu- sion 12 hours and then the mouse (n=8) with the processing of following programs；(3) I/R+ medicines (TEMPOL or indoles-bis- TEMPOs 9r, s) Treatment group：Mouse (n=8) received large bolus injection medicine (TEMPOL 9r, s, 30mg/kg, dissolving before 5 minutes in Reperfu- sion In salt solution), then receive the continuous infusion (TEMPOL or 9r, s, 10mg/kg/hr) of medicine in the whole Reperfu- sion phase.To group (1) animal and in (2) applies saline vehicle alternatives to medication.Anaesthetized by intraperitoneal injection yellow Jackets (80mg/kg) male Property Wistar mouse.In whole experiment, body temperature is maintained 37 DEG C by means of heating cushion.Peritonaeum is entered by midline incision Chamber carries out laparotomy ventrotomy.Small intestine gently on wet gauze, is then divided using micro vessel clamp occlusion superior mesenteric artery 45 Clock, then Reperfu- sion 12 hours.Confirm sufficiently to block according to the pale and pulse free of the mesenteric of small intestine.Again Surgical incision is sutured during perfusion.The mouse of sham-operation is subjected to identical operation plan, but is not subjected to press from both sides dead mesenterium and moves Arteries and veins, and anesthesia is kept during experiment.At each time point specified, 8 mouse are put to death at random from every group；Collect blood Liquid sample, liver, kidney, lung, and the through thickness sample of small intestine are collected, to be further analyzed.By blood sample Centrifuged 10 minutes with 1000g, collect serum and be stored in -80 DEG C.

The measurement of 1.8 MDAs (LPO)

As it was previously stated, the amount of LPO in intestines is determined by MDA method.In short, prepare intestinal tissue homogenate, and with containing The reaction solution of lauryl sodium sulfate, acetic acid solution and thiobarbituricacidα- solution incubates 60 minutes at 95 DEG C.It is cooled to After room temperature, H is added₂O and n-butanol, violent oscillation mixture, then centrifuged 10 minutes in 3000g.Existed using spectrophotometer 535nm measures the absorbance of organic layer, using 1,1,3,3- tetraethoxypropane as internal standard.

The measure of 1.9 serum fingerprints (TNF-α)

The serum levels of proinflammatory cytokine TNF-α are used as the representative of systemic inflammatorome.Use commercial ELISA Assay kit Serum TNF-alpha levels in scheme (Jingmei BioTech Co.Ltd, China) the measure supernatant recommended with manufacturer. Its level is expressed as pg/mL.

1.10 biochemical measurement

The kinetic determination alanine amino reacted using automated biological chemical analyzer (Toshiba, Japan) by Jaffe The serum levels of transferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and kreatinin (Cr).Organize credit Analysis

Take out the terminal ileum band of 10mm length immediately after ischaemic/Reperfu- sion operation is completed and is euthanized.It is logical Cross median sternotomy and obtain lung tissue.Then cryogenic thermostat slicer (Leica CM1850UV clinic cryogenic thermostats are used Device) frozen tissue section (4-5mm) is cut at -30 DEG C, respectively with stained with Hematoxylin-Eosin, the trichrome stain of improvement and NADH is dyed, and is then observed under light field (Olympus BX51).Histopathology assessment research is carried out with double-blind fashion.

1.11 statistical analysis

Starting with Origin programs uses two-way ANOVA then to carry out Scheff é inspections between test group Difference.If it is observed that difference, then compare paired data using Student ' s t-test.Numerical value is expressed as average value ± SE. If p<0.05, then it is assumed that result is significant.

2nd, result

2.1 interior anti-inflammatory activity

We have studied indoles-TEMPO conjugates 9a-s anti-inflammatory using the mouse ear Edema Test method of dimethylbenzene induction Property (table 1).As common inflammation-induced agent, dimethylbenzene can induce the serious water of ear by inducing acute inflammatory reaction Swollen change.The modeling provides quick, sensitive and reliably screening antiinflammatory live test method.

Anti-inflammatory activities of the compound 9a-s of table 1 in the ear oedema mouse that dimethylbenzene induces

Indole ring is that have anti-inflammatory and the important pharmacophoric group of antioxidation.As seen from Figure 1, indole derivatives (7f-s) Anti-inflammatory activity be significantly higher than carrier (CMC) anti-inflammatory activity.It is interesting that TEMPO groups introducing indole derivatives is caused Anti-inflammatory activity significantly improves.In some cases, this trend is significant.For example, compound 9f, i, j, p, r, s (agent Measure as 0.10mmol/kg) effective antiinflammatory is shown as, more than the anti-inflammatory power of control (aspirin).Wherein, indoles-bis- TEMPO 9r, s are maximally effective antiinflammatories, realize that 77-80% inflammation suppresses.Apparently, the TEMPO being present in conjugate Part is more, and inflammation suppresses stronger.

Then, have been made by dose-effect relationship and has been characterized to obtain more detailed pharmacodynamics curve by compound 9f, i, j, p, r, s.Apply Add 0.02,0.05 and 0.10mmol/kg dosage as a result, compound 9f, i, j, p, r, it is anti-that s produces dose-dependant anti-inflammatory Answer (Fig. 2).Maximally effective indoles-dinitrogen oxygen compound (9r, s, dosage=0.02mmol/kg) is better than aspirin.It is assuming that scorching Disease process causes the infringement of downstream anti-oxidative defense, it is presumed that what the improvement of 9a-s anti-inflammatory activities enhanced with TEMPO groups Inoxidizability effect is relevant, and further highlighting less structural change can cause efficacy of drugs significantly to cooperate with improvement.

Influence of the 2.2 indoles-TEMPO conjugates (9f, i, j, p, r, s) to myeloperoxidase (MPO) activity

Myeloperoxidase (MPO) activity is a kind of important marker of inflammation.In the dropsy of ear model of dimethylbenzene induction In, MPO activity is reflected by the neutrophil cell accumulation in the oedema biopsy of mouse ear.It is observed that dimethylbenzene Administration dramatically increase in ear MPO activity.However, even under 0.02mmol/kg dosage, indoles-TEMPO is conjugated It is active (Fig. 3) that thing 9f, i, j, p, r, s can significantly reduce elevated MPO.It is observed that suppress oedema by these compounds Good correlation between MPO activity.Therefore, it is proposed that the mechanism of action of these compounds is probably to lower inflammation group Various cell factors such as IL-1b in knitting, the expression of IL-6 and TNF-α (these results will be reported in more detail in other places).

The evaluation of 2.3 hemorrhagic tendencies

Conventional NSAID is generally related to several significant side effects, such as hemorrhage of gastrointestinal tract, platelet function are damaged And prolonged bleeding time.In order to develop safer antiinflammatory, the indoles TEMPO conjugates of these new synthesis are evaluated to normal The possible adverse effect that stops blooding causes hemorrhage complication to be important.Therefore, we with rodent afterbody Hemorrhagometry with The estimation anti-freezing degree related to our indoles-TEMPO conjugates (9a-s).We adjust dermatorrhagia time experimental program, To assess the anti-coagulation properties of these compounds.As a result, it was confirmed that under high dose (0.50mmol/kg), our Yin Diindyl-TEMPO conjugates (9a-s) do not extend the bleeding time (table 2) of mouse significantly at all assessment time points.

Influence of the indoles-TEMPO conjugates (9a-s) of table 2 to mouse tail bleeding time (X ± SD)

N=10；NS=Sahne.

2.4 molecular dynamics simulations are studied

Compound 7a-s and 9a-s molecular dynamics simulation result are as shown in figure 13.

Based on the above results, it is proposed that indoles-TEMPO conjugates can be infiltrated into by suppressing neutrophil cell Inflammation part plays its anti-inflammatory activity.In this process, the nitrogen oxides part of these compounds can be reduced into azanol, Azanol can be used as effective free radical scavenger (Fig. 4 A).The high accessibility of nitroxide groups and the height of these compounds Effect is relevant.For example, the analog study to indoles-TEMPO conjugates shows, compound 9b nitrogen oxides part is embedded in Between adjacent substituent, and compound 9p nitrogen oxides part protrudes from the remainder of molecule.This can be same tie Both analogs (9b in row：32.92% suppresses；9p：63.14% suppress) between observe anti-inflammatory activity difference provide One saying.

The radicals scavenging effect of antioxidant is related to the molecule and biochemical parameters selected, such as highest occupies Molecular orbit (HOMO) energy, the difference of the formation heat between net charge, and azanol and its free radical.In our current works In work, the significant contribution of the key dissociation enthalpy of nitrogen oxides derivative, shape index and solvent up to surface is investigated.Along These thinkings, HOMO ionizing energy may be used as antioxidant and participate in measuring for radical scavenging activity.Characterize electronics supply The HOMO energy of ability is suitable to represent radicals scavenging efficiency, is because suppressing autoxidizable process may include the transfer of electronics With the engagement of H atom extraction.Higher HOMO energy means that molecule is good electron donor.Because the extraction of hydrogen atom It is related to electronics transfer, so the HOMO compositions of nitrogen oxides can provide for the avtive spot characteristic of its free-radical scavenging activity Qualitatively opinion.As general rule, HOMO energy is higher, and compound activity for oxidation resistance is higher.For example, 7r, S HOMO energy is respectively -9.374eV, -9.485eV.By contrast, the HOMO energy of their homologue, 9r, s difference It is -5.888eV, -5.895eV (Fig. 4 B).In theory, indoles-bis- TEMPO 9r, s should have than their own parent chemical combination Free radical capture current potential higher thing 7r, s.The high HOMO energy of indoles-TEMPO conjugates highlights their electron donor Current potential and coordination ability, show that they may be used as metallic ion coordination agent and the therewith suppression as ionic catalysis oxidizing process Preparation.

Protective effect of the 2.5 indoles-TEMPO conjugates in simulated ischemia/Reperfu- sion (sI/R) cell model

Next, we have examined these compounds closely protects cells from I/R inductions extremely in I/R cell models are simulated The ability died.In our analysis, Human umbilical vein endothelial cells (HUVEC) are carried out in the process of circulation to simulate I/R damages Wound.During simulated ischemia, significant cell death is not observed.Filled again within 3 hours after cell is exposed to simulated ischemia Note, cell death is more than 80%.The phase difference image of these cells shows cellular swelling and the irregular evidence of film during Reperfu- sion (image is not shown).In order to check the effect of indoles-TEMPO derivatives, different Yin is supplemented cell before simulated ischemia Diindyl-TEMPO derivatives 24 hours, then Reperfu- sion 3 hours.Cell survival rate result is shown in Fig. 5.With individually simulation I/R phases Than after indoles-TEMPO derivative precincubation, viable count dramatically increases.However, compared with control cell, only filling again Indoles-TEMPO derivatives are given during note and do not produce significant protection (Fig. 5).We conclude that these compounds are protected The cell of I/R inductions is protected from death.

The mitigation of 2.6 Mitochondrial oxidative damages

In order to monitor active oxygen (ROS) generation between ischemic stage in mitochondria, HUVEC cells are marked with MitoProbe. MitoProbe is a kind of new fluorescent dye, and it is to mitochondria O₂ ^-·Detection be extremely sensitive.Studied by common location, MitoProbe fluorescence and MitoTracker distribution (Fig. 6) are compared using confocal microscope, it was confirmed that MitoProbe's Specificity.During simulated ischemia/Reperfu- sion (sI/R), mitochondria experienced different morphological changes, be divided into three classes： Tubulose (substantially normal), middle (tubulose with swelling area) and fragmentation (small is spherical).Control cell shows hollow wire Plastochondria, and show hypofluorescence caused by MitoProbe (Fig. 6).During simulated ischemia (sI), the display enhancing of some cells Mitochondria fluorescence, MitoProbe fluorescence are limited in the scatternet of the tubular structure around core.On the contrary, simulated again In the HUVEC cells of perfusion, it is observed that more scattered and irregular staining pattern, strong glimmering with MitoProbe Light.The MitoProbe fluorescence of enhancing shows that simulated ischemia (120 minutes) causes mitochondria O₂ ^-·~2 times of level increase.As institute Predict, 3h Reperfu- sions cause mitochondria O after simulated ischemia₂ ^-·5~6 times of level rise.These data highlight increased mitochondria Oxidative stress associates with simulation I/R's.Indoles-bis- TEMPO processing significantly reduces mitochondria O₂ ^-·Level, this can be from MitoProbe fluorescence intensity is found out；Meanwhile the HUVEC cell numbers increase of display tube plastochondria, show described place Reason significantly alleviates oxidativestress damage in the mitochondria of HUVEC cells.

The suppression of 2.7 mitochondrial cytochrome c releases

Cytochrome c be discharged into from intermembrane space in cytosol be mitochondrial apoptosis activation in primary event.Therefore, we Next determine whether indoles-TEMPO conjugates can suppress the release of cytochrome c using immunofluorescence technique.In Baseline bar Under part, the dot pattern of fluorescence is shown to the HUVEC cells of cytochrome c immunostaining.Representational image such as Fig. 7 institutes Show.

During simulated ischemia, the cell for being not detected by the cytochrome c dyeing of display diffusivity dramatically increases.In sI/R Period, mitochondria O₂ ^-·Level dramatically increases, and the cell number of display diffusivity fluorescence mode also dramatically increases.These results show Mitochondrial cytochrome c is discharged into cytosol after I/R is simulated.Next we check indoles-TEMPO conjugates to sI/R The influence of mitochondrial cytochrome c releases afterwards.Low-level line grain is maintained with the cell of indoles-bis- TEMPO 9r, s pretreatments Body O₂ ^-·And cytochrome c is retained in organelle.As shown in fig. 7, indoles-bis- TEMPO 9r, during s significantly inhibits sI/R Cytochrome c release, cell count show, the cell that cytochrome c discharges is down to 29.8 from 71.2% ± 4.5% ± 3.2% (9r) or 23.1% ± 3.0% (9s).These results show the mitochondria O induced by sI/R₂ ^-·Excess generation facilitate Mitochondria fragmentation and cytochrome c release, these are all by aobvious in the cell with indoles-bis- TEMPO 9r, s pretreatments Write and suppress.

2.8 intestinal ischemia-reperfusion in mice damage

Then we direct attention to intestinal tract injury pattern.Gut ischmia-reperfusion (I/R) damage is that ripe whole body is scorching Disease animal model, and multiple organ failure and serious morbid state can be caused.It is excellent due to indoles-bis- TEMPO 9r, s Anti-inflammatory and antioxidation activity, it is intended to which lacking in intestines I/R damage mouse models could further be mitigated by illustrating these compounds Blood-reperfusion injury.

The histologic analysis of 2.9 small intestines

We carry out evaluation structure using various (h and E (H and E)) dyeing and changed；The core of nicotinamide adenine two Thuja acid (NADH) diaphorase is dyed to assess tissue metabolism state；And improved trichrome stain distinguishes cell and connective Tissue, to estimate the histologic effect of indoles-TEMPO analogs.In the mouse group for being subjected to intestines I/R damage models, Wo Menguan Observe the shortening of fine hair, the loss of chorioepithelium and significant mucosal neutrophils infiltration (the top row in Fig. 8 A).It is identical Improved trichrome stain shows various viscous from the stripping of chorioepithelium to obvious extravasated blood (Fig. 8 B top row) in tested mouse Membrane damage, this is also apparent in the tissue (the top row in Fig. 8 C) of NADH dyeing.Abnormal (the contracting of fine hair of these histologies The presence of short, exposed fine hair, mucosal ulcer and crypts) it is the feature that intestines I/R is damaged.It is dry with the double TEMPOs (9r, s) of indoles This tissue damage can significantly be mitigated in advance, the fluff tip only slightly peeled off is shown and preserve good gland structure (Fig. 8 A-C Bottom row).

Intestinal mucosal injury evaluation is as follows：0 grade, normal mucosa, 1 grade：Slight epithelium departs from；2 grades：Moderate epithelium departs from；3 Level：Extensive epithelium departs from；4 grades：Exposed fine hair, 5 grades：Ulcer.

Influence of the 2.10 indoles-TEMPO conjugates (9r, s) to lipid peroxidation

Free radical is formed and effect of the inflammation in intestines I/R damages promotes us to study indoles-TEMPO conjugates in intestines Potentially beneficial effect in ischemia/reperfusion injury.Therefore, we quantify the intestines MDA in mouse tissue (LPO) (Fig. 8 D), this reveals that the horizontal direct relations between the local inflammation reaction order of severity of I/R damages of LPO.With The mouse of sham-operation is compared at identical time point, is being damaged with salt solution+I/R in the mouse of processing, the LPO water in intestinal tissue It is flat to dramatically increase.Occurs the maximum LPO accumulation of 412.4 ± 63.7nmol/μ g tissues after the damage 3 hours of Reperfu- sion induction. On the contrary, before perfusion and applying TEMPOL or 9r, s during continuous pouring, cause in the notable of all testing time point LPO Reduce (Fig. 8 D).Cell membrane phospholipid is also easy to peroxidating during I/R is damaged, and causes LPO to produce.Our research prompting, To give TEMPOL or 9r, s before and during Reperfu- sion to intervene, may cause to greatly reduce LPO being produced in intestines, this shows 9r, S protective value is at least partly derived from its radical scavenging activity.In addition, intestines I/R damages are by stimulating inflammatory cytokine Generation with chemotactic factor (CF) and promote inflammatory reaction.

Effect of the 2.11 indoles-TEMPO conjugates (9r, s) to serum fingerprint (TNF-α) concentration

Because TNF-α is the critical mediator of a part induced by ischemia/reperfusion and long-range organ damage, we Next assess its horizontal (Fig. 8 E).When Reperfu- sion is completed, it is subjected to serum TNF-cc level in the mouse group of intestines I/R damages and shows Writing increase, (62.9 ± 8.7pg/mL contrasts 8.7 ± 2.3pg/mL (sham-operation group, p<0.01).The effect is because using TEMPOL (25.2 ± 5.2pg/mL), 9r (21.7 ± 4.7pg/mL) or 9s (19.0 ± 3.8pg/mL) and mitigate (in all cases p<0.01)。

The histologic analysis of 2.12 nephridial tissues

The serious renal tubular necrosis (Fig. 9 A-C) of intestines I/R wound inducements vacuolation in proximal tubule, including renal tubule expand Open and downright bad, tube chamber extravasated blood caused by the disintegration of tubular structure, and the loss on bristle border.Improvement in identical tissue Trichrome stain show with the infiltration of vascular system red blood cell, the renal tubule oedema in interstitial tissue, cellular infiltration and matrix Expand (Fig. 9 B).In addition, rupture (the figure in Bowman's capsules is observed in the NADH stained tissues for being derived from intestines I/R mouse groups 9C).In nephridial tissue, in the case of no intervention, it is bad to substantially reduce renal tubule with the administration of indoles-bis- TEMPO (9r, s) Extremely.Most of renal tubules are complete, have normal brush border.In general, the histological change in nephridial tissue is quite warm With although see the renal tubule of proximal tubule in the animal groups for applying indoles-bis- TEMPOs (9r, s) with can cutting in and out Expansion and vacuolation.Shown with the trichrome stain evaluation collagenous fibres of improvement, collagen around the renal tubule related to I/R damages Extensive loss weakened by indoles-bis- TEMPO (9r, s).The Mean histology scoring of salt solution group kidney is 8.5 ± 2.5, significantly Higher than sham-operation group (0.60 ± 0.2, p<0.001), and the Mean histology of indoles-bis- TEMPO conjugates scoring (9r, s) is aobvious Writing reduces (4.3 ± 1.7, p<0.01).

The order of severity based on renal damage, including cellular swelling, vacuolation, renal tubular necrosis, cast in renal tubule Into the forfeiture of tubular dilator and brush border, injury of kidney is classified as follows：0 grade：Normal histology；1-2 levels：Minor injury；3~4 Level：Moderate lesion；5~6 grades：Grievous injury.

2.12 indoles-influences of the bis- TEMPOs (9r, s) to blood urea nitrogen (BUN) and creatinine (Cr)

Our shadows by quantitative blood urea nitrogen (BUN) and creatinine (Cr) indirect assessment indoles-bis- TEMPOs to renal function Ring.3 hours after Reperfu- sion, the BUN levels in the mouse group of I/R damages that are subjected to dramatically increase (Fig. 9 D).In indoles-bis- In the animal groups of TEMPOs (9r, s) processing, BUN also increases and reaches peak value for 3 hours after Reperfu- sion, but definitely BUN water It is flat to be substantially less than the level (p for being subjected to being observed in the animal groups of I/R damages<0.01).Change of serum C r levels are also observed comparable As a result (Fig. 9 E).

The histologic analysis of 2.13 hepatic tissues

In hepatic tissue, the Histological change after intestines I/R damages includes oedema, liver cell vacuolation and inflammatory infiltration.Dye Going through for color section shows that serious sinus property is congested, bleeding, thicker central vein, subendothelial oedema and with core week The denaturation liver cell of vacuolation.Intervened with indoles-bis- TEMPOs (9r, s) and mitigate this infringement, the only slight sinus property of display Expansion and central vein are thicker, and most of liver cells seem normal.The histological score of salt solution group mouse liver is significantly big In sham-operation group (p<0.001), indoles-bis- TEMPOs (9r, s) administration significantly reduces the histological score related to intestines I/R Increase.Although liver cell change is still visible, it has been found that indoles-bis- TEMPOs have under intervening to be understood and consistent tissue Learn and improve (Figure 10 A-C).

The classification of severity of liver tissue injury is as follows：0 grade：Normal histology；1 grade：Minor injury, including cell Matter vacuolation is to focal karyopycnosis；2 grades：Moderate has an extensive karyopycnosis to severe injury, and cytoplasm acidophil granules is thin Born of the same parents increase to be lost with iuntercellular border；3 grades：Severe necrosis, crushed with hepatic cell cords, bleeding and neutrophil cell infiltration.

2.14 indoles-bis- TEMPOs (9r, s) are to liver enzyme (ALT (ALT) and aspartate transaminase (AST)) the influence of serum levels

Compared with sham-operation mouse, intestines I/R damages cause the notable rise of Serum ALT and AST levels, are damaged with liver cell Wound is consistent.At all time points tested, intervened with indoles-bis- TEMPO (9r, s) of selection and significantly reduced to serum liver This influence (Figure 10 D and E) of enzyme.

The histologic analysis of 2.15 lung tissues

Our evaluation is transferred to lung tissue from kidney and intestinal tissue, it has been found that I/R damages cause collapsing for lamina propria Burst, bleeding, the exacerbation (Figure 11 A-C) that interstitial cell and lung structure destroy, along with the cellular infiltration in alveolar space.I Find that in pulmonary capillaries and interstitial tissue interstitial thickens and obtained with neutrophil cell infiltration after being intervened with 9s significantly Improve.Although all show the positive, drug therapy through making NADH dyeing to lung from I/R groups and indoles-bis- TEMPOs (9r, s) groups Differential dyeing result in group significantly reduces, and this shows to intervene through indoles-bis- TEMPOs (9r, s), and tissue viability, which has, to be changed It is kind.

Injury of lungs classification is as follows：0 grade：Diagnose unchanged；1 grade：It is slightly congested to moderate interstitial；2 grades：Moderate blood vessel week Enclose the partial injury of oedema formation and lung structure；3 grades：The structure of lung is destroyed completely.

3rd, discuss

The excessive generation of active oxygen (ROS) can promote acute inflammation.In inflammation part, increased free radical produce with it is thermophilic The uncoupling of the activation of neutrophil NADPH oxidase and/or various oxidation-reduction systems is relevant.By strengthening free radical Scavenging activity, which improves intracellular reaction, can promote the maintenance of the balance between ROS and Antioxidative Defense System.In this research In, the combination of indole derivatives 7a-s and nitrogen oxides part enhances the antiinflammatory action of indoles-TEMPO conjugates (9a-s). We further determine that indoles-TEMPO conjugates 9f, i, j, p, r, s can be obviously reduced the otitis of dimethylbenzene inducing mouse Disease, it was demonstrated that its potential anti-inflammatory activity (Fig. 1).These compounds can also significantly inhibit the MPO increased by dimethylbenzene induction Activity.It has been reported that there is direct contact between the increase of MPO activity and neutrophil cell accumulation in Inflamed tissue.Therefore, I Propose the anti-inflammatory activity of these indoles-TEMPO conjugates or be related to neutrophil cell infiltration suppression and leukocyte movement Reduction.In inflammation part, activation and/or various oxygen of the increased free radical activity with neutrophil cell nadph oxidase The uncoupling that original system is gone back in change is relevant.Inflammation typically results in the damage of Antioxidative Defense System.Therefore, we further assume that Improvement of the indoles-TEMPO conjugates newly synthesized to anti-inflammatory activity can be with the radical scavenging activity phase of nitrogen oxides part Close.

It is reported that TEMPOL can be by suppressing transition metal iron catalytic action scavenger-cell in superoxide anion And prevent from forming hydroxyl radical free radical.The clinical effect of the platelet function imbalance of NSAIDs inductions includes bleeding increase, persistently There is serious or threat to life bleeding risk and increased in operative hemorrhage and patient.In order to develop safer antiinflammatory, me is assessed New indole-TEMPO conjugates in normal hemostasis with the presence or absence of can cause to tend to having for hemorrhage complication It is critical that evil, which influences,.In all testing time points, with our new anti-inflammatory agent (9a-s) of high dose (0.50mmol/kg) Treatment does not extend the bleeding time (table 1) of mouse significantly.Obviously, the indoles-TEMPO conjugates phase of these new synthesis with Control drug aspirin relatively shows good pharmacological characteristic.

In view of the extensive anti-inflammatory of compound (9r, s) and antioxidation activity, we then characterize select indoles Effect of-TEMPOL compounds the kind (9r, s) in intestines I/R injured animal models, comprehensive assessment intestines, liver kidney, blood and Antioxidative Defense System (table 3).The extensive injuries of the intestines of I/R wound inducements, it is characterised in that universal intestinal submucosa tissue damage The mark of wound, exposed fine hair, lamina propria disintegration, the infiltration of exposed capillary and neutrophil leucocyte/macrophage be present (Figure 10 A-C).Using compound 9r, s significantly reduces this damage.Nephridial tissue performance from the animal in I/R damage groups Go out extensive renal tubular necrosis, plus with cast in the vacuolation in proximal tubule and renal tubule into.These histological changes It is denatured including tubular structure, nephric tubule expansion, protein fragments, swelling, necrosis and intracavitary extravasated blood, all symptoms are with brush The loss (Figure 11 A-C) of shape edge.In addition, 1.5,3 and 12 hours after I/R, using TEMPOL or 9r, I/R damages are obviously reduced The rise of caused blood urea nitrogen.

In hepatic tissue, I/R damages cause serious sinus property congested and bleeding, with the increase with the vacuolation of core week Liver cell (Figure 10 A-C).The histologic analysis of hepatic tissue shows cell death and inflammatory infiltration in I/R treatment groups, with change It is mitigated again after compound 9r, s processing.In addition, on hepatic tissue, it has been found that the horizontal of AST and ALT damages in I/R Apply TEMPOL or 9r, s postnormalize within 1.5,3 and 12 hours afterwards.

The histologic analysis of lung tissue shows the serious alveolar collapse in the mouse of I/R groups, and intra-alveolar hemorrhage and alveolar are several What shape degenerates.These histological changes much less in 9r, s+I/R groups.We have found that indoles-bis- TEMPOs 9r, s show The seriousness for mitigating ALI is write, and suppresses the Apoptosis of lung after intestines I/R damages.

The indoles of table 3.-bis- TEMPOs (9r, s) intervene different caused by the mouse for being subjected to intestine ischemia/reperfusion injury Effect

System	The result of measurement	Indoles-bis- TEMPOs
			Intestines	Structure, form, LPO	++ (improvement)
Liver	Structure, form, AST, ALT	++ (improvement)
			Kidney	Structure, form, BUN, Cr	++ (improvement)
Lung	Structure, form	++ (improvement)
			Anti-oxidative defense	LPO, TNF-, cell factor, free radical	+++ (improvement)

Our notice is turned into anti-oxidative defense, it has been found that in the intestinal tissue of mouse of I/R damages is subjected to LPO is dramatically increased, and significantly improves this change (table 3) using TEMPOL or indoles-bis- TEMPO (9r, s), and this most has can It can be caused by radicals scavenging.When we evaluate the generation of inflammatory cytokine and chemotactic factor (CF), it was found that similar knot Fruit.Our result of study show to be shown as by the inflammation damnification of I/R wound inducements it is systemic because TNF-α (participates in I/R damages The main inflammatory mediators of wound) sharply increase by being improved using TEMPOL or indoles-bis- TEMPO (9r, s).

In the research of antioxidation activity of the indoles-TEMPO conjugates in I/R cell models, it has been found that simulation The cell death of I/R inductions can be weakened by these compounds.Also, these compounds can be obviously reduced mitochondrial oxidation Damage.In addition, these compounds can substantially reduce the mitochondrial cytochrome c releases as caused by simulation I/R.However, we These compounds are not illustrated also reduces the precise mechanism that mitochondrial cytochrome c is discharged into cytosol.Indoles TEMPO is conjugated The free radical scavenging activity of thing can be produced to contribute to this protective effect by suppressing mitochondria ROS.The indoles proposed- TEMPO conjugates and the mechanism of cytochrome c interaction are shown in fig. 12.Another possibility is that these compounds can To modify Bcl-2 family members protein expression or function, to be responsible for keeping mitochondrial membrane integrality.

In our current researchs, mouse with indoles-TEMPO mouse treat, receive within 5 minutes before Reperfu- sion indoles- TEMPO's injects, and then receives indoles TEMPO continuous infusion during whole Reperfu- sion.In similar research, lacking Therapeutic treatment is always applied before the blood phase.Our therapeutic strategy may be suitable for occurring the excessive risk of gastrointestinal complication Patient.This therapeutic choice is can contemplate especially for the patient of the operation of experience abdominal aorta and openheart surgery.At present, We do not know whether still protected when indoles-TEMPO conjugates are applied after Reperfu- sion.Because proinflammatory factor is in intestine ischemia Played a significant role during Reperfu- sion, thus indoles-TEMPO conjugates dual-use function (antioxidant and anti-inflammatory activity and Local and remote organ improvement) even if reperfusion injury should be able to be alleviated after injury by applying.Current at us In research, mesenteric I/R complication clinically is simulated using I/R serious model.

, in the indoles-TEMPO of continuous infusion low dosage, (can it be existed in some clinical indications with rational prediction Under less serious ischemia injury) preferably protection will be obtained.

The content of above example only to illustrate the invention, but should not be construed as limiting the invention.Without departing substantially from this In the case of spirit and essence, the modifications or substitutions made to the inventive method, step or condition belong to the present invention Scope.

Claims

1. a kind of indoles-TEMPO conjugates, it is characterised in that there is the structure shown in formula I：

Wherein, X is carbon, oxygen, nitrogen, sulphur, silicon, phosphorus or boron atom；X is combined by saturation or unsaturated bond；X can be with any substitution Base forms key；N is selected from 4-6 integer；

Wherein, R is selected from the C of hydrogen, substituted or unsubstituted, straight chain, ring-type or side chain_1-5Alkyl or carbonyl, amide groups or substituted The cycloalkyl that amide groups or R collectively form the cycloalkyl of nitrogen atom or are substituted with N atoms.

2. indoles-TEMPO conjugates as claimed in claim 1, it is characterised in that in formula I,Group is selected from

3. indoles-TEMPO conjugates as claimed in claim 1, it is characterised in that described substituted or unsubstituted, straight chain, The C of ring-type or side chain_1-5Alkyl or carbonyl are included through amino, 3- nitros guanidine radicals, imidazole radicals, phenyl, hydroxy phenyl, alkylthio group, hydroxyl Base or the C of indyl substitution_1-5Alkyl or carbonyl, described substituted amide groups include 1- epoxides -2,2,6,6- tetramethyl piperazines Pyridine -4- bases) the methyl substituted formamido of -1,2,3- triazole-4-yls or acetamido.

4. indoles-TEMPO conjugates as claimed in claim 1, it is characterised in that R is selected from hydrogen, methyl, isopropyl, positive fourth Base -2- bases, isobutyl group, 2- amino -2- oxoethyls, imidazol-4 yl-methyl, benzyl, 4- aminobutyls, para hydroxybenzene first Base, 2-methylmercaptoethyl, methylol, indol-3-yl-methyl, R1 and N atoms are collectively forming pyrroles -2, N- base, 1- hydroxyl second Base,3- amino 3- oxopropyls,

5. indoles-TEMPO conjugates as claimed in claim 1, it is characterised in that described indoles-TEMPO conjugates are selected from Compound with following structure：

6. it is a kind of synthesize claim any one of 1-5 described in indoles-TEMPO conjugates method, it is characterised in that including with Lower step：

Since optical activity L-Trp, parent B-carboline 1 is prepared by Pictect-Spengler reactions, then protected The coupling of shield property, obtains intermediate 4 and 5；In the 3- positions of B-carboline ring, different amino acid is introduced to produce a series of indole derivativeses 7a-s；Then, terminal acetylene functional group is introduced as synthesis " handle " to introduce TEMPO parts, and nitrine is catalyzed by copper (I) Compound/alkynes cycloaddition reaction obtains required object 9a-s：

Wherein, reagent and condition are：I formaldehyde and sulfuric acid；IIBoc2O and triethylamine；III benzylalcohols, polyphosphoric acid, backflow；IV HOBt, DCC；V H₂, Pd/C；VI L-AA-OBzl, HOBt, DCC；VII HCl/EtOAc；VIII H₂, Pd/C；Propargylamine, HBTU, triethylamine；IX 4- azidos-TEMPO, CuI.

7. indoles-TEMPO the conjugates described in claim any one of 1-5 are far apart organ confrontation ischemia-reperfusion in preparation protection Purposes in damage medicine.