CN106047959B - Alpha -chymotrypsin is synthesizing application and method in double indyl-Indolin-2-one class compounds - Google Patents
Alpha -chymotrypsin is synthesizing application and method in double indyl-Indolin-2-one class compounds Download PDFInfo
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Abstract
The invention discloses the Friedel-Crafts reactions of alpha -chymotrypsin catalysis to synthesize application and method in double indyl-Indolin-2-one class compounds, general formula 1 and general formula 2 can be generated to double indyl-Indolin-2-one class compounds under the catalysis of alpha -chymotrypsin in methanol or ethanol solution, the catalytic process of this method is simple to operation, yield reaches as high as 97%, show splendid substrate adaptability, not only enrich the multi-functional research of enzyme, also the synthesis for double indyl-Indolin-2-one class compounds provides an efficient route of synthesis.
Description
Technical field
The invention belongs to chemical field, the Friedel-Crafts reaction for being related to alpha -chymotrypsin catalysis is synthesizing double indyls-two
Application in hydrogen indoles -2- ketone compounds, the method for further relating to prepare double indyl-Indolin-2-one class compounds.
Background technique
Indoles is because its special structure feature since 1866 are found, has had become including medicine, fragrance, agricultural
The research hotspot of the numerous areas such as chemicals, pigment and material science, these work clearly demonstrate indoles in organic synthesis
Middle occupied critical role.
Bisindole structure is present in many drugs.This analog derivative is proven to have extensive bioactivity, than
Antiviral such as antiplasmodial, antibacterial is antitumor, anticonvulsion, the treatment of parkinsonism and effective sars coronavirus 3CL albumen
Enzyme inhibitor.Wherein, double indyl-Indolin-2-ones, which are found to be a kind of, has significant anti-inflammatory, AntiHIV1 RT activity, anti-tumor activity
Substance.The a series of bis- indyl indolones of 3,3- are found to have anticancer, antiviral activity.What is interesting is, it was reported that
Certain double indyl-Indolin-2-one derivatives can consumingly act on a series of tumour cell, but will not be directed to
Normal cell.Double indyls-extensive use of the Indolin-2-one skeleton in pharmaceutical chemistry causes chemists great
Interest to develop the route of synthesis of highly effective, it has been reported that method in, such heterocyclic synthesis most straightforward approach is
Three component tandem reactions between catalysis isatin and indoles obtain corresponding target product.
Alpha -chymotrypsin (α-Chymotrypsin from bovine pancreas) is also referred to as chymotrypsin,
Be to extract the endopeptidase isolated from the fresh pancreas of ox, be a kind of serine protease, can will with aromatic hydrocarbon or its
The peptide that his large-scale hydrophobic side chains (Tyr, Trp, Phe, Met, Leu) combine is hydrolyzed from the c-terminus of key.It is studied as one kind
Very clear hydrolase, the stereochemical structure of alpha -chymotrypsin has been elucidated with, the list being made of 241 amino acid residues
One peptide chain includes 5 pairs of disulfide bond in molecule.The molecular weight of alpha -chymotrypsin is about 25KDa, optimal pH 8-9, activity
Site is the 57th histidine, the 102nd aspartic acid and the 195th serine, is made of these three active sites
Catalytic triads structure play a part of catalytic center.
In the technique study that synthesis has natural products similar structures, target product one is synthesized with simple and direct efficient method
It has been the interest place of scientists study since straight.Therefore, the synthesis for double indyl-Indolin-2-one class compounds mentions
For an efficient route of synthesis, also the multi-functional research of horn of plenty enzyme is very important.
Summary of the invention
In view of this, one of the objects of the present invention is to provide alpha -chymotrypsins in Fu for being catalyzed general formula 1 and general formula 2
Gram reaction generates the application in double indyl-Indolin-2-one class compounds；The second object of the present invention is to provide preparation
Double indyls-Indolin-2-one class compound method.
For achieving the above object, the invention provides the following technical scheme:
2,1, alpha -chymotrypsin is catalyzed general formula 1 in methanol solution or ethanol solution and the Friedel-Crafts reaction of general formula 2 is raw
Application in indyl in pairs-Indolin-2-one class compound:
R1Optionally H or methyl；R2Optionally H, F, Cl, Br, nitro, methyl or methoxy；R3Optionally H or methyl；R4
Optionally H, F, Br, methyl or methoxy.
Preferably, the R1Optionally H or methyl；R2Optionally H, F, Br, nitro or methoxyl group；R3For H；R4Optionally
H, F, methyl or methoxy.
Preferably, the R1Optionally H or methyl；R2Optionally H, F or methoxyl group；R3For H；R4Optionally H, F, methyl
Or methoxyl group.
2, the method for preparing double indyl-Indolin-2-one class compounds, includes the following steps:
It is substrate with general formula 1, general formula 2, alpha -chymotrypsin is catalyst, and methanol solution or ethanol solution are solvent,
It is stirred to react at 25~30 DEG C, double indyl-Indolin-2-one class compounds is made；
R1Optionally H or methyl；R2Optionally H, F, Cl, Br, nitro, methyl or methoxy；R3Optionally H or methyl；R4
Optionally H, F, Br, methyl or methoxy.
Preferably, the R1Optionally H or methyl；R2Optionally H, F, Br, nitro or methoxyl group；R3For H；R4Optionally
H, F, methyl or methoxy.
Preferably, the R1Optionally H or methyl；R2Optionally H, F or methoxyl group；R3For H；R4Optionally H, F, methyl
Or methoxyl group.
Preferably, the methanol solution or ethanol solution are the solution of 15~30% water containing volume fraction.
Preferably, the molar ratio of the general formula 1 and general formula 2 is 1:2.
Preferably, the enzyme amount of alpha -chymotrypsin is 0.93~1.87kU.
Preferably, double indyl-Indolin-2-one class compounds are prepared and monitor reaction process in the process, reaction is completed
After filter out alpha -chymotrypsin, washed with ethyl acetate, be dissolved in product all in filtrate, filtered with anhydrous sodium sulfate is dry
Filtrate is concentrated liquid, and concentrate obtains target by flash column chromatography as eluent using ethyl acetate/petroleum ether and produces
Object.
The beneficial effects of the present invention are: alpha -chymotrypsin can be urged as a kind of environmentally protective biocatalyst
Change the reaction between isatin (and its derivative) and indoles (and its derivative), is synthesized in methanol solvate by Friedel-Crafts reaction
Double indyls-Indolin-2-one class compound, have obtained very high yield (yield reaches as high as 97%), and show
Splendid substrate adaptability.This method expands application of the alpha -chymotrypsin in catalytic chemistry conversion, not only enriches
The multi-functional research of enzyme also provides an efficient conjunction for the synthesis of double indyls-Indolin-2-one class compound
At approach.The catalytic process of this method is simple to operation, is the double indyl-Indolin-2-one class compounds of organic synthesis
A kind of practical approach.
Detailed description of the invention:
Fig. 1 be alpha -chymotrypsin catalysis Friedel-Crafts reaction time course (reaction condition: isatin (0.30mmol),
Indoles (0.60mmol), deionized water (0.2mL), methanol (0.8mL), alpha -chymotrypsin (0.93kU, solid powder)
It is stirred to react under the conditions of 30 DEG C.Yield obtains after being separated by silica gel column chromatography).
Fig. 2 be isatin and 2 methyl indole Friedel-Crafts reaction (aReaction condition: isatin (0.30mmol), 2 methyl indole
(0.60 mmol), deionized water (0.2mL), methanol (0.8mL), alpha -chymotrypsin (0.93kU, solid powder) is at 30 DEG C
Under the conditions of be stirred to react 60h；bYield obtains after being separated by silica gel column chromatography).
Fig. 3 is the comparative experiments that alpha -chymotrypsin is catalyzed Friedel-Crafts reaction.
Fig. 4 is that alpha -chymotrypsin is catalyzed the possible mechanism of Friedel-Crafts reaction.
Fig. 5 is that alpha -chymotrypsin catalysis general formula 1 reacts the double indyl-Indolin-2-one classes of generation with general formula 2
Close object reaction equation.
Specific embodiment
It is described in detail below in conjunction with preferred embodiment.Test method without specific conditions in embodiment,
Usually according to conventional conditions or according to the manufacturer's recommendations.
One, the Friedel-Crafts reaction of alpha -chymotrypsin catalysis synthesizes the screening of the solvent of double indyl-Indolin-2-ones
It is carried out using the reaction of compound 1a (isatin) and compound 2a (indoles) in different solvents as model reaction
α-chymotrypsin catalytic effect in different solvents is probed into, and is compared to 17 kinds of different solvents, experimental result
It is shown in Table 1.
When selection solvent such as dimethyl sulfoxide, n,N-Dimethylformamide this kind of polar aprotic solvent (table 1, serial number
1,2) when, the generation of reaction is not observed.And in methylene chloride, 1,2- dichloroethanes (table 1, serial number 14,15)
The activity of reaction significantly improves, and has obtained 73% yield.(table 1, serial number in polar protic solvent methanol and ethyl alcohol
16,17) product that double indoles replace is generated to reaction selectivity.Based on this discovery, this solvent control product is generated anti-
It should be studied respectively.In conclusion selection 1,2- dichloroethanes aoxidizes Yin as monosubstituted Friedel-Crafts reaction synthesis 3- hydroxyl
The solvent of diindyl derivative, and be that disubstituted Friedel-Crafts reaction synthesizes the molten of double indyl-Indolin-2-one class compounds with methanol
Agent.Experimental result is shown in Table 1.
The screening of 1 solvent of tablea
aReaction condition: isatin (0.30mmol), indoles (0.45mmol), deionized water (0.1mL), solvent (0.9mL),
Alpha -chymotrypsin (0.93kU, solid powder) is stirred to react under the conditions of 25 DEG C.bYield is separated by silica gel column chromatography
After obtain.
Two, the influence for the Friedel-Crafts reaction that water content is catalyzed alpha -chymotrypsin
It is non-specific in a large amount of enzymatic before it was found that, the addition of suitable quantity of water can be largely in organic solvent
Ground influences the effect of enzymic catalytic reaction, and therefore, the content for having studied different water in methyl alcohol is catalyzed indigo to alpha -chymotrypsin
The influence (table 2) of the red Friedel-Crafts reaction between indoles.It is isatin in substrate molar ratio: in the case where indoles=1:2, with containing
Water increases to 20% (table 2, serial number 1-5) from 0, and the yield of disubstituted product increases rapidly to very excellent 93%, and is continuing
The yield reacted when increasing water content starts to reduce (table 2, serial number 6-10).Therefore, 20% is confirmed as the reaction most
Good water content.
The influence for the Friedel-Crafts reaction that 2 water content of table is catalyzed alpha -chymotrypsina
aReaction condition: isatin (0.30mmol), indoles (0.60mmol), deionized water (0-0.60mL), methanol (1.0-
0.4mL), alpha -chymotrypsin (0.93kU, solid powder) is stirred to react 72h under the conditions of 25 DEG C.bYield passes through silicagel column
It is obtained after chromatography.
Three, the influence for the Friedel-Crafts reaction that enzyme amount is catalyzed alpha -chymotrypsin
In order to maximally utilise the catalytic efficiency of catalyst, the enzyme amount of reaction system is screened, to make
This model reaction is catalyzed with least alpha -chymotrypsin.From the structure of disubstituted reaction product, we are not difficult to find out reaction
It is to occur between the isatin of monovalent and the indoles of two equivalents, because isatin and indoles molar ratio have obtained when being 1:2
Quite satisfied yield has been arrived, has just been examining for best substrate molar ratio progress enzyme amount with this ratio the considerations of for atom economy
Examine (table 3).After a series of experiment for having carried out enzyme amount we have seen that enzyme amount be 0.93kU when obtained best 93%
Yield (table 3, serial number 3).So selecting the enzyme amount of 0.93kU best for the double indoles substitution reactions of alpha -chymotrypsin catalysis
Catalytic amount.
The influence for the Friedel-Crafts reaction that 3 enzyme amount of table is catalyzed alpha -chymotrypsina
aReaction condition: isatin (0.30mmol), indoles (0.60mmol), deionized water (0.2mL), methanol (0.8mL),
Alpha -chymotrypsin (0.31-2.18kU, solid powder) is stirred to react 72h under the conditions of 25 DEG C.bYield passes through silicagel column
It is obtained after chromatography.
Four, the influence for the Friedel-Crafts reaction that temperature is catalyzed alpha -chymotrypsin
In view of temperature for the importance of enzymic catalytic reaction, double indoles that temperature is catalyzed alpha -chymotrypsin are had studied
The influence (table 4) of substitution reaction.It has all been obtained not under the conditions of these temperature 15 DEG C to 30 DEG C from can see in table 4
Wrong yield (table 4, serial number 1-4), wherein having obtained optimal effect when 30 DEG C, yield has reached 96% (table 4, sequence
Number 4).And when temperature continues to increase, yield starts slowly to decline, and has a decline rapidly at 60 DEG C, drops to only
8% (table 4, serial number 7).To sum up as a result, selected 30 DEG C are the reaction most suitable reaction temperature.
The influence for the Friedel-Crafts reaction that 4 temperature of table is catalyzed alpha -chymotrypsina
aReaction condition: isatin (0.30mmol), indoles (0.60mmol), deionized water (0.2mL), methanol (0.8mL),
Alpha -chymotrypsin (0.93kU, solid powder) is stirred to react 72h in different temperatures.bYield is separated by silica gel column chromatography
After obtain.
Five, the time course of the Friedel-Crafts reaction of alpha -chymotrypsin catalysis
After the optimization that the model reaction being catalyzed to alpha -chymotrypsin has carried out series reaction condition, and then exist
The time course of model reaction is explored under optimal conditions.It further studies alpha -chymotrypsin and is catalyzed double indoles
The time course (Fig. 1) of substitution reaction.The initial stage started as can be drawn from Figure 1 in reaction, the yield of disubstituted product
Rapidly rise, at first 72 hours of reaction, yield increased as time increases, and reached highest when 72 hours
96%, extend the reaction time again later, yield does not continue growing.
Six, the control experiment of the Friedel-Crafts reaction of alpha -chymotrypsin catalysis
It in order to verify the disubstituted reaction is catalyzed by alpha -chymotrypsin, has done a series of control experiments (table 5).
When replacing alpha -chymotrypsin catalysis reaction with bovine serum albumin and egg white powder, 29% and 16% has been respectively obtained
(table 5, serial number 12 and 13) illustrate what reaction was mainly catalyzed by alpha -chymotrypsin to yield, and it is non-specific for eliminating
The possibility of the protein surface amino acid residue catalytic of property.Then still enzyme has been carried out 2 hours with urea and guanidine hydrochloride respectively
Pretreatment has only been respectively obtained 24% and 56% yield by the disubstituted reaction of urea and the processed enzymatic of guanidine hydrochloride
(table 5, serial number 2 and 4).And at the same time urea be individually catalyzed with guanidine hydrochloride this react respectively obtain 12% and 40% yield
(table 5, serial number 3 and 5) show that the enzyme activity crossed by Urea treatment only remains 9.3U/mg after the measurement for having done enzyme activity to it, and
17.2U/mg is also dropped to by the processed enzyme activity of guanidine hydrochloride.Because urea and guanidine hydrochloride can be with the three-dimensional space knots of destructive enzyme
Structure, it is possible to it is vital to think that the three-dimensional structure of enzyme plays the role of in being catalyzed the reaction.Next respectively to work
Three different amino acid residues in property site have carried out inhibition processing.Alpha -chymotrypsin has been carried out 2 hours with DCC
When being catalyzed the reaction after processing, it has been found that the yield of target product falls below 14% (table 5, serial number 6), and individually DCC is catalyzed
The disubstituted product (table 5, serial number 7) of trace is only obtained.And 44% receipts are obtained by the processed enzymic catalytic reaction of DPEC
Rate (table 5, serial number 8), but DPEC itself can be catalyzed to obtain 30% disubstituted product (table 5, serial number 9).By enzyme activity
Measurement we have found that by the processed enzyme activity of DPEC only remain 8.3U/mg.Finally, being handled with PMSF enzyme, equally
Good effect has been obtained, 46% yield (table 5, serial number 10) is obtained by the processed enzymatic of PMSF at this time, and PMSF is mono-
Can solely be catalyzed to obtain 40% yield (table 5, serial number 11), to being carried out after enzyme activity determination by the processed enzyme of PMSF we have found that
Its vigor only has 4.4U/mg.In conclusion may be considered alpha -chymotrypsin be catalyzed it is disubstituted between isatin and indoles
Reaction.
The control experiment of the Friedel-Crafts reaction of 5 alpha -chymotrypsin of table catalysisa
aReaction condition: isatin (0.30mmol), indoles (0.60mmol), deionized water (0.2mL), methanol (0.8mL),
Alpha -chymotrypsin (0.93kU, solid powder) is stirred to react under the conditions of 30 DEG C.bYield is separated by silica gel column chromatography
After obtain.cVigor definition: it is 25 DEG C in temperature, under conditions of pH is 7.8, hydrolyzes 1 μm of ol N- benzoyl-L- junket per minute
The enzyme amount of propylhomoserin ethyl ester is 1U.d480mg urea (is dissolved in 1mL deionized water by the urea liquid that the enzyme of 0.93kU is dissolved in 8M
In) at room temperature remove water by freeze-drying after stir process 2 hours.eThe enzyme of 0.93kU is dissolved in the guanidine hydrochloride solution of 6mM
(573mg guanidine hydrochloride is dissolved in 1mL deionized water) is at room temperature removed water by freeze-drying after stir process 2 hours.fIt will
The enzyme of 0.93kU is dissolved in the DCC solution (206mg DCC is dissolved in 1mL deionized water) of 1mM, and stir process 2 is small at room temperature
When after by water by freeze-drying remove.g43.4 μ L DEPC (are dissolved in 1mL by the DEPC solution that the enzyme of 0.93kU is dissolved in 0.3mM
In the phosphate buffer that pH is 8.04) water is removed by freeze-drying after stir process 2 hours at room temperature.hBy the enzyme of 0.93kU
The PMSF solution (105mg PMSF is dissolved in 1mL tetrahydrofuran) for being dissolved in 0.6mM at room temperature will after stir process 2 hours
Organic solvent is spin-dried for.
Seven, the substrate extension of the Friedel-Crafts reaction of alpha -chymotrypsin catalysis
After having carried out series of optimum to the condition of reaction, in order to verify alpha -chymotrypsin to this type of catalysis
The substrate adaptability of reaction has investigated the reaction between a variety of different substituents and the isatin and indoles of different the position of substitution.Such as
Shown in table 6 and Fig. 2, alpha -chymotrypsin has obtained corresponding a variety of different substitutions as an efficient catalyst
3,3-, bis- indyl oxidation diketone product, and obtain good yield.When 1 of isatin by it is methyl substituted when with
Indoles with different substituents all shows good reactivity worth (table 6, serial number 4,13 and 14).When 5 of isatin is inhale
When electron substituent group (table 6, serial number 3,7,12,15,17 and 18), mostly with 5 indoles with electrophilic and supplied for electronic
It shows well to arrive outstanding reaction effect, wherein (21) table 6 and has obtained extraordinary yield at serial number 3,12,15,19.When
5 of isatin have obtained extraordinary reaction effect (table 6, serial number 11 and 23) when being electron-donating group.It should be noted that
After being replaced by methyl for 1 of indoles, relatively poor reactivity (table 6, serial number 5- are all shown with different types of isatin
7,16,20 and 22).It is anti-by 72 hours in addition, we extend disubstituted Friedel-Crafts reaction of the isatin with 2 methyl indole
Ying Hou has obtained 85% yield.The yield of comprehensive substrate, alpha -chymotrypsin is to the disubstituted Fu Ke occurred in methyl alcohol
Reaction shows very outstanding catalytic capability.
The substrate extension of the Friedel-Crafts reaction of 6 alpha -chymotrypsin of table catalysisa
aReaction condition: 1 (0.30mmol), 2 (0.60mmol), deionized water (0.2mL), methanol (0.8mL), α-pancreas are solidifying
Galactase (0.93kU, solid powder) is stirred to react under the conditions of 30 DEG C.bYield obtains after being separated by silica gel column chromatography.
Eight, the comparative experiments and possible mechanism of alpha -chymotrypsin catalysis Friedel-Crafts reaction
In order to determine, whether enzyme takes part in reaction in the second step for generating disubstituted reaction, has done pair as shown in Figure 3
Than experiment.At 30 DEG C, the mixing that mono-substituted product 3a and indoles are added to methanol and water with the ratio of molar ratio 1:1 is molten
It in agent, is then compared respectively with addition enzyme with enzyme is added without, as a result, it has been found that the case where no enzyme and other catalyst are added
Under, disubstituted 4a can not be spontaneously generated, and we have obtained 97% yield in the reaction that enzyme is added, it is possible thereby to
Conclude that enzyme has also assisted in reaction in the substitution of second step indyl.
After a series of control experiments that alpha -chymotrypsin takes part in every single step reaction and front have been determined, base
In the activated centre of alpha -chymotrypsin, we have proposed Fu Ke between an alpha -chymotrypsin catalysis isatin and indoles is anti-
Answer possible catalytic mechanism.As shown in figure 4, firstly, 3 carbonyls are by Asp- in alpha -chymotrypsin active site in isatin
His-Ser catalysis three is coupled 195 serines activation in structures, the carbonyl then protonated by the indoles attack of electron rich,
The intermediate for forming an enamine cation, sloughs proton in serine under the action of negative oxygen ion, obtain mono-substituted oxygen
Change indoles.In polar protic solvent methanol, hydroxyl is seized by Labile protons and generates a molecular water in mono-substituted product, so
After generate imines ion, by intermolecular imines enamine interconversion, obtained a new enamine cation, then another molecule
Indoles attack this intermediate, form new enamine cation, seized in intermediate again by negative oxygen ion in serine
Proton finally falls off to obtain the Oxoindole that final double indyls replace from alpha -chymotrypsin.
Nine, the Friedel-Crafts reaction of alpha -chymotrypsin catalysis generates the reality of double indyl-Indolin-2-one class compounds
It tests
Alpha -chymotrypsin catalysis general formula 1 is obtained according to above-mentioned optimal conditions, and the double indyls-two of generation are reacted with general formula 2
Hydrogen indoles -2- ketone compounds (Fig. 5), optimum condition are as follows: 0.3mmol general formula 1 is sequentially added in the round-bottomed flask of 10mL
Compound, the compound of 0.6mmol general formula 2,0.8mL methanol, 0.2mL deionized water, 0.93kU alpha -chymotrypsin,
It is stirred to react at 30 DEG C, the monitoring reacted with thin-layer chromatography.After reaction, enzyme is filtered out, is washed with ethyl acetate
Filter cake is dissolved in product all in filtrate, then uses anhydrous sodium sulfate dried filtrate, and crude product is obtained after filtrate is concentrated, and then will
Crude product obtains disubstituted target product (petroleum ether: ethyl acetate=1:1-3:1) by flash column chromatography.
The product of acquisition is detected through hydrogen nuclear magnetic resonance spectral method, and data are as follows:
Compound (4a):
1H NMR(600MHz,DMSO-d6) δ 10.95 (s, 2H), 10.59 (s, 1H), 7.37 (d, J=7.7Hz, 2H),
7.25-7.24 (m, 4H), 7.03-6.99 (m, 3H), 6.96-6.93 (m, 1H), 6.87 (s, 2H), 6.82-6.80 (t, J=
6.8&6.7,2H).13C NMR(150MHz,DMSO-d6)δ179.30,141.85,137.47,135.16,128.31,126.26,
125.41,124.80, 121.95,121.44,121.30,118.74,114.90,112.10,110.08,53.14.
Compound (4b):
1H NMR(600MHz,DMSO-d6) δ 11.02 (s, 2H), 10.68 (s, 1H), 7.39 (d, J=8.1Hz, 2H),
7.27 (d, J=8.1Hz, 2H), 7.11-6.98 (m, 5H), 6.94 (d, J=2.2Hz, 2H), 6.84 (t, J=7.6Hz, 2H)
.13C NMR(150 MHz,DMSO-d6)δ179.21,159.18,157.61,137.49,136.91,126.10,124.94,
121.54,121.12,118.89, 114.68,114.58,114.25,113.00,112.84,112.19,110.86,53.67.
Compound (4c):
1H NMR(600MHz,DMSO-d6) δ 11.34 (s, 1H), 11.10 (s, 2H), 8.25 (dd, J=8.7,2.3Hz,
1H), 8.02 (d, J=2.2Hz, 1H), 7.41 (d, J=8.2Hz, 2H), 7.24 (dd, J=19.4,8.4Hz, 3H), 7.06
(t, J=7.6Hz, 2H), 7.00 (d, J=2.5Hz, 2H), 6.86 (t, J=7.6Hz, 2H)13C NMR(150MHz,DMSO-
d6)δ179.39,148.30, 142.74,137.55,135.81,125.91,125.10,121.69,120.82,120.58,
119.07,113.37,112.34,110.39, 53.08.
Compound (4d):
1H NMR(600MHz,DMSO-d6) δ 11.03 (s, 2H), 7.42 (d, J=8.1Hz, 2H), 7.37 (dd, J=
16.6,7.8Hz, 2H), 7.26 (d, J=8.1Hz, 2H), 7.19 (d, J=7.8Hz, 1H), 7.07 (dd, J=14.2,
7.1Hz, 3H), 6.93 (d, J=2.4Hz, 2H), 6.86 (t, J=7.6Hz, 2H), 3.31 (s, 3H)13C NMR(150MHz,
DMSO-d6)δ177.43, 143.26,137.48,134.24,128.45,126.17,125.08,124.83,122.62,
121.46,121.20,118.80,114.64, 112.11,109.09,52.70,26.71.
Compound (4e):
1H NMR(600MHz,DMSO-d6) δ 10.59 (s, 1H), 7.37 (d, J=8.3Hz, 2H), 7.25-7.20 (m,
4H), 7.08 (t, J=7.6Hz, 2H), 6.98 (d, J=7.7Hz, 1H), 6.92 (t, J=7.5Hz, 1H), 6.88 (s, 2H),
6.83 (t, J=7.5Hz, 2H), 3.70 (s, 6H)13C NMR(150MHz,DMSO-d6)δ179.00,141.76,137.83,
134.99,128.94,128.35, 126.53,125.36,122.01,121.47,118.86(s),113.99,110.14,
52.89,32.80.
Compound (4f):
1H NMR(600MHz,DMSO-d6) δ 10.44 (s, 1H), 7.36 (d, J=8.3Hz, 2H), 7.26 (d, J=
8.1Hz, 2H), 7.08 (t, J=7.6Hz, 2H), 6.91 (d, J=7.6Hz, 3H), 6.82 (ddd, J=12.1,10.9,
5.0Hz,4H),3.70(s, 6H),3.60(s,3H).13C NMR(150MHz,DMSO-d6)δ178.88,155.19,137.85,
136.33,135.14, 129.00,126.54,121.49,118.87,113.98,112.66,110.37,110.20,55.80,
53.37,32.80.
Compound (4g):
1H NMR(600MHz,DMSO-d6) δ 10.83 (s, 1H), 7.44 (dd, J=8.3,1.8Hz, 1H), 7.42-7.35
(m, 3H), 7.29 (d, J=8.1Hz, 2H), 7.12 (t, J=7.6Hz, 2H), 7.01 (d, J=8.3Hz, 1H), 6.97 (s,
2H), 6.89 (t, J=7.5Hz, 2H), 3.70 (s, 6H)13C NMR(150MHz,DMSO-d6)δ178.58,141.16,
137.90,137.39, 131.26,129.05,127.89,126.39,121.72,121.23,119.10,113.79,
113.26,112.22,110.36,53.19, 32.82.
Compound (4h):
1H NMR(600MHz,DMSO-d6) δ 10.88 (d, J=18.3Hz, 2H), 10.56 (s, 1H), 7.31-7.16 (m,
4H), 6.99 (d, J=7.7Hz, 1H), 6.96-6.85 (m, 3H), 6.76 (d, J=8.1Hz, 1H), 6.71-6.61 (m, 2H),
6.53 (d, J=8.1Hz, 1H), 2.12 (s, 3H), 1.99 (s, 3H)13C NMR(150MHz,DMSO-d6)δ179.83,
141.75, 136.11,135.50,134.42,132.53,128.25,127.63,125.97,121.72,120.25,
120.22,120.13,118.43, 110.98,110.82,109.93,52.99,13.66,13.48.
Compound (4i):
1H NMR(600MHz,DMSO-d6) δ 11.16 (s, 2H), 10.74 (s, 1H), 7.42 (dd, J=8.8,4.7Hz,
2H), 7.28 (dd, J=9.8,7.8Hz, 2H), 7.07 (d, J=7.7Hz, 1H), 7.04 (d, J=2.5Hz, 2H), 7.02-
6.89(m,5H). 13C NMR(150MHz,DMSO-d6)δ179.08,157.54,156.01,141.79,134.43,134.20,
128.58,126.80, 126.35,125.39,122.21,114.81,113.16,110.29,109.90,109.73,
105.74,105.58,52.79.
Compound (4j):
1H NMR(600MHz,DMSO-d6)δ10.79(s,2H),10.58(s,1H),7.26–7.20(m,4H),7.00(d,
J=7.7 Hz, 1H), 6.94 (t, J=7.5Hz, 1H), 6.86 (s, 2H), 6.72-6.67 (m, 4H), 3.52 (s, 6H)13C
NMR(150 MHz,DMSO-d6)δ179.23,152.94,141.93,135.05,132.69,128.28,126.63,125.63,
125.44,121.94, 114.17,112.51,110.93,109.91,103.90,55.64,53.02.
Compound (4k):
1H NMR(600MHz,DMSO-d6)δ11.29(s,2H),10.81(s,1H),7.47–7.42(m,4H),7.30(t,
J=7.6 Hz, 1H), 7.25-7.21 (m, 3H), 7.10 (d, J=7.7Hz, 1H), 7.03-7.01 (m, 3H)13C NMR
(150MHz, DMSO-d6)δ179.01,141.74,136.25,134.15,128.74,127.84,126.51,125.38,
124.18,123.24, 122.31,114.37,111.66,110.38,52.74.
Compound (4l):
1H NMR(600MHz,DMSO-d6) δ 11.21 (s, 2H), 10.77 (s, 1H), 7.42 (dt, J=22.1,11.1Hz,
2H), 7.12 (t, J=8.9Hz, 2H), 7.06 (d, J=12.2Hz, 3H), 6.94 (dd, J=15.0,8.9Hz, 4H)13C
NMR(150MHz, DMSO-d6)δ179.00,159.29,157.65,156.06,137.99,136.08,134.19,126.92,
126.17,115.07, 114.92,114.16,113.29,113.22,112.97,111.12,110.00,109.83,
105.59,105.43,53.29.
Compound (4m):
1H NMR(600MHz,DMSO-d6) δ 10.89 (s, 2H), 10.81 (s, 1H), 7.44 (dd, J=8.3,1.6Hz,
1H), 7.32 (d, J=8.9Hz, 3H), 7.01 (d, J=8.3Hz, 1H), 6.95 (d, J=2.3Hz, 2H), 6.76 (dd, J=
8.8,2.1Hz,2H), 6.71(s,2H),3.56(s,6H).13C NMR(150MHz,DMSO-d6)δ178.85,153.16,
141.33,137.43, 132.77,131.18,127.99,126.47,125.83,113.69,113.43,112.77,
112.08,111.14,103.69,55.72, 53.33.
Compound (4n):
1H NMR(600MHz,DMSO-d6) δ 10.94 (s, 2H), 7.39 (d, J=8.3Hz, 4H), 7.17 (d, J=
6.9Hz, 1H), 7.06 (d, J=27.3Hz, 3H), 6.82 (d, J=15.3Hz, 4H), 3.61 (s, 6H), 3.33 (s, 3H)13C
NMR(150MHz, DMSO-d6)δ177.63,153.18,143.42,134.25,132.84,128.54,126.68,125.80,
125.25,122.77, 114.10,112.72,111.26,108.97,103.73,55.63,52.77,26.63.
Compound (4o):
1H NMR(600MHz,DMSO-d6) δ 11.22 (s, 2H), 7.45 (dd, J=9.1,4.4Hz, 2H), 7.36 (dd, J
=11.0,7.6Hz, 2H), 7.17 (d, J=7.7Hz, 1H), 7.06 (d, J=8.5Hz, 3H), 6.94 (d, J=8.7Hz,
4H),3.30(s, 3H).13C NMR(150MHz,DMSO-d6)δ177.23,157.60,156.07,143.19,134.24,
133.58,128.71, 126.85,126.34,125.07,122.89,114.61,113.23,109.99,109.81,
109.29,105.70,105.54,52.40, 26.71.
Compound (4p):
1H NMR(600MHz,DMSO-d6) δ 10.92 (s, 2H), 10.75 (s, 1H), 7.35 (d, J=8.5Hz, 2H),
7.16-7.05 (m, 3H), 7.01 (d, J=1.9Hz, 2H), 6.79 (d, J=8.7Hz, 4H), 3.60 (s, 6H)13C NMR
(150MHz, DMSO-d6)δ179.34,159.26,157.69,153.16,138.21,136.84,132.81,126.56,
125.84,114.79, 114.64,113.63,113.04,112.88,112.72,111.12,110.82,103.85,55.70,
53.67.
Compound (4q):
1H NMR(600MHz,DMSO-d6) δ 10.78 (s, 1H), 7.39 (ddd, J=10.7,8.0,3.2Hz, 4H),
7.19-7.11 (m, 4H), 7.11-7.05 (m, 1H), 7.03 (d, J=3.6Hz, 2H), 6.93 (td, J=7.4,3.8Hz,
2H),3.72(s,6H). 13C NMR(150MHz,DMSO-d6)δ179.04,159.25,157.68,138.09,137.93,
136.78,129.14,126.50, 121.72,121.36,119.10,114.87,114.72,113.47,113.08,
112.92,110.96,110.31,53.54,32.79.
Compound (4r):
1H NMR(600MHz,DMSO-d6) δ 11.43 (s, 1H), 11.29 (s, 2H), 8.27 (dd, J=8.7,2.0Hz,
1H), 8.04 (s, 1H), 7.44 (dd, J=9.2,4.6Hz, 2H), 7.26 (d, J=8.7Hz, 1H), 7.14 (d, J=2.0Hz,
2H), 6.94 (t, J=8.5Hz, 4H)13C NMR(150MHz,DMSO-d6)δ179.20,157.65,156.12,148.15,
142.95,135.07, 134.27,127.10,126.02,120.64,113.64,113.26,110.59,110.20,
110.02,105.34,105.18,52.71.
Compound (4s):
1H NMR(600MHz,DMSO-d6) δ 11.39 (s, 1H), 10.98 (s, 2H), 8.27 (dd, J=8.6,1.8Hz,
1H), 8.07 (d, J=1.5Hz, 1H), 7.34 (d, J=8.7Hz, 2H), 7.25 (d, J=8.6Hz, 1H), 7.02 (d, J=
2.2Hz, 2H), 6.77 (d, J=8.8Hz, 2H), 6.74 (s, 2H), 3.58 (s, 6H)13C NMR(150MHz,DMSO-d6)δ
179.43,153.24, 148.36,142.77,135.75,132.84,126.36,125.91,120.60,112.89,
112.70,111.28,110.30,103.46, 55.70,53.04.
Compound (4t):
1H NMR(600MHz,DMSO-d6) δ 11.24 (s, 2H), 10.92 (s, 1H), 7.46 (dd, J=8.2,4.4Hz,
2H), 7.34 (d, J=8.0Hz, 1H), 7.30 (s, 1H), 7.10 (d, J=8.5Hz, 3H), 7.00 (d, J=10.2Hz, 2H),
6.95 (t, J=8.6 Hz, 2H)13C NMR(150MHz,DMSO-d6)δ178.80,157.66,156.13,140.74,
136.40,134.24,128.61, 126.97,126.36,126.19,125.26,114.07,113.34,111.85,
110.00,109.91,105.59,105.43,53.10.
Compound (4u):
1H NMR(600MHz,DMSO-d6) δ 10.82 (s, 1H), 7.41 (t, J=12.3Hz, 2H), 7.31 (dd, J=
15.1,8.3 Hz, 3H), 7.26 (s, 1H), 7.12 (t, J=7.5Hz, 2H), 7.06 (d, J=8.3Hz, 1H), 6.98 (s,
2H), 6.90 (t, J=7.4 Hz, 2H), 3.71 (s, 6H)13C NMR(150MHz,DMSO-d6)δ178.70,140.75,
137.90,137.01,129.05, 128.40,126.40,126.08,125.21,121.71,121.24,119.10,
113.26,111.66,110.35,53.23,32.82.
Compound (4v):
1H NMR(600MHz,DMSO-d6) δ 11.19 (s, 2H), 10.85 (s, 1H), 7.47 (dd, J=8.3,1.7Hz,
1H), 7.41 (dt, J=14.3,7.2Hz, 2H), 7.33 (d, J=1.4Hz, 1H), 7.02 (d, J=7.9Hz, 3H), 6.95-
6.85(m,4H). 13C NMR(150MHz,DMSO-d6)δ178.51,157.55,156.02,141.11,136.70,134.16,
131.45,127.86, 126.90,126.07,114.08,113.31,112.35,110.02,109.85,105.46,
105.30,52.94.
Compound (4w):
1H NMR(600MHz,DMSO-d6) δ 11.37 (s, 1H), 8.27 (dd, J=8.7,2.2Hz, 1H), 8.04 (d, J=
2.0Hz, 1H), 7.40 (dd, J=17.5,9.9Hz, 2H), 7.30 (t, J=7.5Hz, 2H), 7.23 (d, J=8.7Hz, 1H),
7.12 (t, J=7.7Hz, 2H), 7.04 (s, 2H), 6.90 (t, J=7.6Hz, 2H), 3.73 (s, 6H)13C NMR(150MHz,
DMSO-d6)δ 179.18,148.23,142.79,137.96,135.66,129.24,126.26,125.95,121.82,
121.02,120.65,119.24, 112.45,110.46,52.89,32.85.
Compound (4x):
1H NMR(600MHz,DMSO-d6) δ 10.87 (s, 2H), 10.55 (s, 1H), 7.36 (d, J=8.7Hz, 2H),
7.02 (d, J=7.2Hz, 3H), 6.93-6.86 (m, 2H), 6.85 (s, 2H), 6.80 (d, J=8.6Hz, 2H), 3.64 (d, J
=12.1Hz, 3H), 3.60 (s, 6H)13C NMR(150MHz,DMSO-d6)δ179.31,155.30,153.10,136.47,
135.41,132.83, 126.73,125.81,114.26,113.00,111.03,110.35,104.07,55.76,53.63.
Compound (4z):
1H NMR(600MHz,DMSO-d6) δ 10.87 (d, J=22.8Hz, 2H), 10.71 (s, 1H), 7.36 (d, J=
8.3Hz, 2H), 7.33 (d, J=7.4Hz, 1H), 7.29 (t, J=7.5Hz, 1H), 7.22 (s, 2H), 7.11 (d, J=
7.7Hz, 1H), 7.00 (t, J=7.4Hz, 1H), 6.94 (t, J=9.8Hz, 4H), 2.31 (s, 6H)13C NMR(150MHz,
DMSO-d6)δ179.58, 141.94,136.00,135.28,128.29,127.00,126.55,125.52,125.12,
123.18,121.96,121.01,114.46, 111.90,110.07,53.28,21.99.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (10)
1. alpha -chymotrypsin is catalyzed general formula 1 in methanol solution or ethanol solution and the Friedel-Crafts reaction of general formula 2 generates double Yin
Application in diindyl base-Indolin-2-one class compound:
R1Optionally H or methyl；R2Optionally H, F, Cl, Br, nitro, methyl or methoxy；R3Optionally H or methyl；R4Optionally
For H, F, Br, methyl or methoxy.
2. application according to claim 1, it is characterised in that: the R1Optionally H or methyl；R2Optionally H, F, Br, nitre
Base or methoxyl group；R3For H；R4Optionally H, F, methyl or methoxy.
3. application according to claim 1, it is characterised in that: the R1Optionally H or methyl；R2Optionally H, F or methoxy
Base；R3For H；R4Optionally H, F, methyl or methoxy.
4. the method for preparing double indyl-Indolin-2-one class compounds, which comprises the steps of:
It is substrate with general formula 1, general formula 2, alpha -chymotrypsin is catalyst, and methanol solution or ethanol solution are solvent, 25
It is stirred to react at~30 DEG C, double indyl-Indolin-2-one class compounds is made；
R1Optionally H or methyl；R2Optionally H, F, Cl, Br, nitro, methyl or methoxy；R3Optionally H or methyl；R4Optionally
For H, F, Br, methyl or methoxy.
5. according to the method described in claim 4, it is characterized by: the R1Optionally H or methyl；R2Optionally H, F, Br, nitre
Base or methoxyl group；R3For H；R4Optionally H, F, methyl or methoxy.
6. according to the method described in claim 4, it is characterized by: the R1Optionally H or methyl；R2Optionally H, F or methoxy
Base；R3For H；R4Optionally H, F, methyl or methoxy.
7. according to the method described in claim 4, it is characterized by: the methanol solution or ethanol solution are containing volume fraction 15
The solution of~30% water.
8. according to the method described in claim 4, it is characterized by: the molar ratio of the general formula 1 and general formula 2 is 1:2.
9. according to the method described in claim 4, it is characterized by: the enzyme amount of alpha -chymotrypsin is 0.93~1.87kU.
10. according to the method described in claim 4, it is characterized by: preparing double indyl-Indolin-2-one class compounds
Reaction process is monitored in the process, filters out alpha -chymotrypsin after the reaction was completed, is washed with ethyl acetate, keeps product completely molten
In filtrate, with anhydrous sodium sulfate dried filtrate, filtrate is concentrated, concentrate passes through by eluent of ethyl acetate/petroleum ether
Flash column chromatography obtains target product.
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