CN1194006A - Conversion of indene to (IS) -amino- (2R) -indanol free of any stereoisomer using a combination of dioxygenase biotransformation and chemical steps - Google Patents

Abstract

A process is disclosed for the bioconversion of indene to (1S) -amino- (2R) -indanol substantially free of any stereoisomer by the action of a dioxygenase enzyme followed by several chemical steps, e.g., chiral specific crystallization, treatment with a strong acid in the presence of acetonitrile.

Description

Utilize the conversion of indenes that the dioxygenase bio-transformation combines with chemical step to (1S) of free of any stereoisomer-amino-(2R)-indanol

Background of invention

The U.S.S.N.08/059 that the application and Merck submitted at May 7 in 18996,1993,038, the U.S.S.N.08/235 that submits to of 18996IA, on April 29th, 1994,576, Merck19251, Merck 19114, Merck 19115 and 19351 relevant.

The present invention relates to a kind of synthetic can HIV inhibiting (HIV) compound of encoded protein enzyme, especially as the method for the intermediate of some the oligopeptides analogues of the compound J among the following embodiment.The prevention that these compounds infect HIV, the treatment of treatment that HIV infects and the acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)) that is caused by HIV has value.These compounds also can be used for suppressing feritin and some other proteolytic enzyme.

Invention described here is to (2S)-amino-(1R) conversion of indanol, shown in following scheme I, II and III about indenes.

Scheme I Scheme II Scheme III

A kind of retrovirus of having named to human immunodeficiency virus (HIV) is to comprise carrying out property of immunity system damage (acquired immune deficiency syndrome (AIDS); Acquired immune deficiency syndrome (AIDS)) and maincenter and peripheral nervous system degenerate at the virulence factor of interior complex disease.This virus once was called LAV, HTLV-III or ARV.The universals that retrovirus duplicates are the precursor polyprotein is carried out a large amount of translation post-treatment generation virus by the proteolytic enzyme of encoding viral assembling and the necessary ripe viral protein of function.The inhibition of this course of processing can stop the generation that is generally infective virus.For example, Kohl, N E etc., Proc.Natl Acad.Sci.85, the genetic inactivation of 4686 (1988) middle proof HIV encoded protein enzymes has caused immature, as not have infectious virion generation.These results show that the inhibition of hiv protease can be used as a kind of feasible method that treatment acquired immune deficiency syndrome (AIDS) and prevention or treatment HIV infect.

The nucleotide sequence of HIV demonstrates and have a pol gene (Ratner, L etc., Nature, 313,277 (1985)) in an open reading frame.The homology of aminoacid sequence provides the evidence (Toh, H. etc., EMBO J.4,1267 (1985) of pol sequence encoding ThermoScript II, a kind of endonuclease and a kind of hiv protease; Power, M.D. etc., Science, 231,1567 (1986); Pearl, L.H. etc., Nature, 329,351 (1987)).The end product compound that comprises some oligopeptides analogue that can be made by new intermediate of the present invention and method is the inhibitor of hiv protease, and is disclosed in disclosed EPO 541,168 on May 12nd, 1993.Referring to for example wherein compound J, this compound also illustrates among the embodiment below.

The application discloses the modification method of following formula 1 (S)-amino-2 (R)-hydroxy indenes that a kind of preparation is essentially steric isomer purity:

This structure is the side-chain radical of compound J, and compound J is the potent inhibitor of hiv protease.

It is lower to produce the efficient of raceme 1 (+/-)-amino-2 (+/-) hydroxy indene from racemic indene oxide in the synthetic trial in the past.In other the synthetic trial with the fungi haloperoxidase with the indenes bio-transformation obtain dominant trans-(2S, 1S)-the bromine indanol, with after various chemical step is translated into (1S)-amino-(2R)-indanol.Also have some other synthetic trial to relate to turn into to be used as intermediate steps and to carry out chemosynthesis, split with L-tartrate subsequently with racemation epoxy.

The invention provides the improvement alternative method that needs.In the method for the invention, by the substrate indenes is generated cis (1S, 2R)-the stereoselectivity biological oxidation of indane glycol with subsequently utilize the special crystallization technique of chirality to being essentially the pure cis of steric isomer-(1S, 2R)-indane glycol crystalline separates and to combine, and saved the tartrate splitting step.Further chemical treatment can obtain (1S)-amino-(2R)-indane glycol, for example be reflected at aqueous acids and exist down and handle with nitrile by Ritter, subsequently again with gegenion to extracting or use the cation-exchange chromatography purifying.

In the method for the invention, by the effect of dioxygenase indenes is converted into and mainly contains required (1S, 2R) mixture of the cis indane glycol of steric isomer.Required (1S, 2R) stereoisomerism body and function purification step separates, for example absorption, extracting, crystallization, with produce pure basically crystallization cis (1S, 2R)-the indane glycol.

That enantiomeric excess surpasses is about 99% (after the special crystallization of chirality from indenes to pure basically cis-(1S, 2R)-indane glycol) be the characteristic feature of the inventive method.

Summary of the invention

The invention provides the biotransformation synthesizing cis that utilizes dioxygenase-(1S, 2R)-novel method of indenes end glycol.Express available toluene in the born of the same parents of dioxygenase and induce (tod), or expression can be non-toluene dependent form in its born of the same parents.Chemical step subsequently can form (1S)-amino-(2R)-indanol, i.e. another intermediate.These product compounds are the intermediates of compound (as compound J) that are used for the inhibitor of synthetic hiv protease, feritin and other proteolytic enzyme.Detailed Description Of The Invention

The invention relates to the method for the intermediate that synthesizes the compound that suppresses hiv protease.Required intermediate is (1S)-amino-(2R) indanol, and it is substantially free of unwanted steric isomer.Another required intermediate be (1S, 2R)-the indane glycol, it be substantially free of unwanted steric isomer (1R, 2S)-the indane glycol.

In the present invention, described synthetic (1S, 2R)-method of indane glycol may further comprise the steps:

A) a certain amount of tod is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) obtain (1S, 2R)-the indane glycol.

Described synthetic be substantially free of any stereoisomer (1S, 2R)-the another kind of method of indane glycol may further comprise the steps:

A) a certain amount of tod is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) purifying (1S, 2R)-the indane glycol;

D) product to step c) carries out the special crystallization of chirality;

E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol.

Described synthetic (1S, 2R)-the another kind of method of indane glycol may further comprise the steps:

A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) obtain (1S, 2R)-the indane glycol.

Described synthetic be substantially free of any stereoisomer (1S, 2R)-the another kind of method of indane glycol may further comprise the steps:

A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) purifying (1S, 2R)-the indane glycol;

D) product to step c) carries out the special crystallization of chirality;

E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol.

The another kind of method of described synthetic (1S) that is substantially free of any stereoisomer-amino-(2R)-indanol may further comprise the steps:

A) a certain amount of tod is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) purifying (1S, 2R)-the indane glycol;

D) product to step c) carries out the special crystallization of chirality;

E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol;

F) in excessive acetonitrile the step e) of dissolving monovalent (1S 2R)-the indane glycol, obtains second kind of mixture, and second kind of mixture maintained approximately between-40 ℃ to about 25 ℃;

G) sneak into excessive normal strong acid, and reaction solution is maintained approximately between-40 ℃ to about 25 ℃;

H) obtain being substantially free of (1S) of any stereoisomer-amino-(2R)-indanol.

The another kind of method of described synthetic (1S) that is substantially free of any stereoisomer-amino-(2R)-indanol may further comprise the steps:

A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

B) fermentation gained mixture;

C) purifying (1S, 2R)-the indane glycol;

D) product to step c) carries out the special crystallization of chirality;

E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol;

F) in excessive acetonitrile the step e) of dissolving monovalent (1S 2R)-the indane glycol, obtains second kind of mixture, and second kind of mixture maintained approximately between-40 ℃ to about 25 ℃;

G) sneak into excessive normal strong acid, and reaction solution is maintained approximately between-40 ℃ to about 25 ℃;

H) obtain being substantially free of (1S) of any stereoisomer-amino-(2R)-indanol.

The biological fractionation effect of described utilization from cis-(1S, 2R)-indane two pure and mild cis-(1R, 2S)-remove in the mixture of indane glycol cis-(1R, 2S)-the another kind of method of indane glycol may further comprise the steps:

A) with a certain amount of dihydrodiol desaturase and cis-(1S, 2R)-indane two pure and mild cis-(1R, 2S)-the mixture contact of indane glycol;

B) fermentation gained mixture;

C) obtain being substantially free of any stereoisomer cis-(1R, 2S)-cis of indane glycol-(1S, 2R)-the indane glycol.

Also having described pseudomonasputida 421-5 is the pure growth of ATCC 55687.

(1S)-amino-(2R) being further purified of indanol can be finished extracting or cation-exchange chromatography by gegenion.

A preferred source of tod is pseudomonasputida F1, and this bacterial strain is preserved in ATCC, and preserving number is 55688.

A preferred source of the dioxygenase of non-toluene dependent form or dioxygenase is pseudomonasputida 421-5, and this bacterial strain is preserved in ATCC, and preserving number is 55687.

The preferred source of dihydrodiol desaturase is pseudomonasputida F1, and this bacterial strain is preserved in ATCC, and preserving number is 55688; Or pseudomonasputida 421-5, this bacterial strain is preserved in ATCC, and preserving number is 55687.

Indenes to cis-(1S, 2R)-indane two pure and mild cis-(1R, 2S)-bio-transformation of indane diol mixture in, any enzyme with bishydroxy effect all is fit to.Preferred enzyme comprises that those can obtain that (1S, 2R) the excessive greatly enzyme of steric isomer is as the dioxygenase (being also referred to as tod) of toluene dependent form or the dioxygenase of non-toluene dependent form.The applicant has isolated a kind of non-toluene dependent form mutant strain pseudomonasputida bacterial strain 421-5, and it can the constitutive expression dioxygenase.Such bacterial strain can save the toluene of enzyme and induce step.

The applicant also finds to have taken place simultaneously the biological fractionation of stereoselectivity in the fermentation of pseudomonasputida and indenes.Under the effect of dihydrodiol desaturase, the unwanted steric isomer cis of this biological fractionation effect degradable-(1R, 2S)-the indane glycol.Therefore impel required steric isomer cis-(1S, 2R)-indane glycol excessive.Any can degrade specifically cis-(1R, 2S)-enzyme of indane glycol is suitable for all that stereoselectivity is biological to be split.

The fermentative action of indenes and the microorganism that contains dioxygenase and optional dihydrodiol desaturase indenes by storage liquid such as silicone oil in the presence of carry out.The oil measure liquid of indenes can be avoided the toxicity of the indenes pair cell of high density.The preferred microorganism that is used to ferment is a pseudomonasputida, most preferably ATCC55688 and 55687.Other microorganism that is fit to comprises the intestinal bacteria with the gene transformation of relevant enzyme, and these genes comprise the gene of pseudomonasputida tod and the pseudomonasputida dihydrodiol desaturase of choosing wantonly.

After the fermentation, with fermenting mixture rotation or centrifugally obtain three layers, the upper strata is an oil measure liquid, is generally silicone oil; The middle level is the water layer that contains required indane glycol bioconversion product; Bottom is cell and residue.The middle level is further processed.

Subsequently the middle level water is mixed to combine the indane diol product with hydrophobic resin.The hydrophobic resin that is fit to includes but not limited to SDEB styrene diethylenebenzene resin, anti-phase C18 resin or uncharged acrylic resin.

Behind water or other the suitable aqueous solvent washing resin, with eluting solvent wash-out resin to remove bonded indane glycol.Eluting solvent comprises any organic solvent, the mixed solution of preferred acetonitrile and water, and most preferred is the mixed solution of about 20% (v/v) acetonitrile and about 80% (v/v) water.Eluting solvent must unmixing with the special crystalline extraction solvent of usefulness chirality.

The indane glycol that wash-out goes out generally carries out the special crystallization of chirality after concentrating.In this process, add extraction solvent, mix with indane glycol in the eluting solvent, keep the extraction solvent layer then.Through the special crystallization of chirality, in the extraction solvent layer the follow-up removal of water with crystallization go out cis-(1S, 2R)-the indane glycol.Suitable extraction solvent comprises isopropyl acetate, toluene, many other organic solvents perhaps, preferred isopropyl acetate.After extraction solvent adds, with the vacuum concentration method except that anhydrating.

The special crystallization of chirality can be used for substituting that stereoselectivity is biological splits or combine with it, with strengthen the required product cis that is substantially free of any stereoisomer-(1S, 2R)-steric isomer of indane glycol is excessive.The ATCC preservation

Submit to a few days ago in U.S. of the application, the sample of microorganism MB 5612 and MB 5652 has been preserved in U.S. typical case's culture collection institute (Rockville, MD 20852 for ATCC, 12301 Parklawn Drive).Culture collection number is respectively ATCC 55688 and 55687.This preservation will be kept 30 years at least at ATCC, and provide to the public after the license that discloses this preservation.The availability that should be pointed out that the preservation thing is not formed under the Patent right condition that infringement government authorizes and implements permission of the present invention.ATCC 55688 and 55687 general feature

Physical features and taxonomy comprise that morphological specificity, cultural characteristic, biological property and physiological characteristic are summarized as follows:

MB 5612-(bacterial strain F1) ATCC 55688: this culture is received with the continuous transfer bodily form formula of pseudomonasputida DSM 6899.It is move about, oxidase positive, gram negative bacillus, 0.38 * 0.76mm, two ends be smooth, be oval.It observes good in fabulous growth on Trypticase soy agar (TSA), eosin methylene blue agar medium (EMB), MacConkey ' s nutrient agar, sheep blood agar culture-medium and Sha Shi maltose nutrient agar (SMA) in 28 and 37 ℃.On TSB bacterium colony transparent, swell and have a complete edge.The bacterium colony quality is butteriness and surperficial floodlight.Do not observe diffusible pigment.Septic smell is arranged.Culture is γ-haemolysis type on blood agar culture-medium.Observed biochemical reaction is as follows on APIRapid NFT plate: the arginine dihydrolase positive; To nitrate, tryptophane, glucose fermentation, urase, and Vitamin C2, gelatin and p-nitrophenyl-β-D-galactopyranoside be hydrolyzed to feminine gender.Oxidation to glucose, seminose (48h), glucose acid, caprate, malate, Citrate trianion and phenylacetate is utilized as the positive.When according to guidance above-mentioned feature being evaluated, the most probable qualification result of this organism is pseudomonasputida (p=0.998).Utilize gas liquid chromatography that whole-cell fatty acid (methyl esters) is analyzed (FAME analysis) and show that its fat composition is consistent with the fat composition of gram negative bacterium, be rich in undersaturated, monounsaturated and cyclopropane fatty acid.Also found the hydroxy fatty acid of low agricultural degree.This organic color atlas and MIDI database are compared the excellent match (Ver 3.6 for similar life=0.913, MIDI Clinical Library) that obtains to pseudomonasputida biotype A.

MB 5652-(bacterial strain 421-5) ATCC 55687: this culture is to receive from MB5612 deutero-strain isolated form.On dull and stereotyped (TSA), observe two kinds of bacterium colony types.A kind of bacterium colony of big brilliant white has central authorities and a kind of less filemot mutation of transparent edge and protuberance, and it is from transparent to opaque, central uplift, and the edge is transparent.Two kinds of bacterium colony mutation also can be observed on other substratum.As if the gramstaining of two kinds of bacterium colony types shows that the cell in the macrocolony is a gram negative bacillus, and 0.76 * 1.5-3.4 μ m, the cell of small colonies also are Gram-negative, but expand on width and variable (0.76-1.14 * 1.9-3.4 μ m).Two kinds of bacterium colony mutation have consistent in fact biochemical character with its parent strain (MB5612) on API Rapid NFT plate.The FAME feature of two kinds of bacterium colony mutation is basically identical each other, but different with its parent strain.Parent strain has 17: 0 cyclopropane (about 35%) of the concentration that raises slightly and reduces by 16: 1 ω 7c (about 11%) of concentration slightly.But these observed differences do not cause two kinds of bacterium colony mutation to be accredited as pseudomonasputida biotype A (similarity of big or small colony type is respectively 0.885 and 0.895).The general remark of culture condition

Preferred carbon source in the nutritional medium is carbohydrate such as glucose, wood sugar, semi-lactosi, glycerine, starch, dextrin etc.Other carbon source that may comprise is maltose, rhamnosyl, raffinose, pectinose, seminose, saligenin, sodium succinate etc.

Preferred nitrogenous source is yeast extract, meat extract, peptone, gluten powder, cottonseed meal, soyflour and other Vegetable powder (part or full degreasing), casein hydrolysate, soybean hydrolyzate and yeast hydrolyate, corn steep liquor, dry yeast, wheat germ, feather meal, peanut powder, vinasse etc., and inorganic and organic nitrogen compound, as ammonium salt (as ammonium nitrate, ammonium sulfate, ammonium phosphate etc.), urea, amino acid etc.

Carbon source and nitrogenous source use favourablely although unite, and needn't use its purified form, because it is also suitable to contain the material than low-purity of micro-growth factor and a great deal of mineral nutrition.If desired, can in substratum, add mineral salt, as yellow soda ash or lime carbonate, sodium phosphate or potassiumphosphate, sodium-chlor or Repone K, sodium iodide or potassiumiodide, magnesium salts, mantoquita, cobalt salt etc.If desired, especially foam when serious, can add defoamer, as whiteruss, fatty oil, vegetables oil, mineral oil or siloxanes at substratum.

As for the condition of mass production cell, preferred deep layer aerobic culture condition, for limited production, can be in shaking bottle wave and culture.And, when in big jar, cultivating, need usefulness-20-70 ℃ of frozen organism of elder generation on substratum relatively in a small amount, inoculating, and go up to cultivate to obtain this organic inoculum at this postvaccinal substratum (being also referred to as " seed culture medium "), be transferred in big jar the inoculum that obtains is aseptic then.The fermention medium that produces inoculum needed autoclaving usually before inoculation.Before autoclaving, the pH of substratum transfers to about 7.0 usually.

The stirring of culturing mixt can realize by many approach with ventilation.Stirring can be by impeller or similar mechanical stirring device, shake a bottle bio-reactor, provide by substratum by different pumping units or by sterile air.Ventilation can be by realizing by sterile air in fermenting mixture.

Fermentation usually between about 25 ℃～37 ℃, preferred 30 ℃, carried out about 0.5～5 day, preferred 2 days, these will change according to fermentation condition and scale.Preferably, produce culture in the bio-reactor that stirs, in about 2 days of 30 ℃ of cultivations, wherein the speed of reactor impeller was about 300rpm.

The used preferred cultivation/production substratum that ferments comprises following substratum: the substratum of table 1. pseudomonasputida F1 bio-transformation indenes is formed (Finette etc., Journalof Bacteriology 160,1004 (1984)).Content during all numerical value are every liter.The material seed culture medium is produced substratum L-arginase 12 .00g 4.00g (NH ₄) ₂SO ₄1.00g 2.00gNa ₂HPO ₄5.24g 10.48gKH ₂PO ₄2.77g 5.54gMgSO ₄-7H ₂O---0.58gCaCl ₂-2H ₂O---0.13g (NH ₄) ₆Mo ₇O ₂₄-4H ₂O---0.36mgFeSO ₄-7H ₂O--the improved Hutners mineral substrate of-3.96mg ^*20mL---improved metal 44 ^*--2.00mL silicone oil--200mL defoamer---1.00mL transfers pH to 7.0 with 6M sulfuric acid ^*Improved Hutners mineral substrate: EDTA, 10g; MgSO ₄-7H ₂O, 14.45g; CaCl ₂-2H ₂O, 3.33g; (NH ₄) ₆Mo ₇O ₂₄-4H ₂O, 0.009g; FeSO ₄-7H ₂O, 0.099g; Metal 44,50mL; Adding distil water to 1 liter; Transfer pH to 6.6-6.8 with 6M sulfuric acid. ^*Improved metal 44 (mg/100mL): EDTA, 250; ZnSO ₄-7H ₂O, 1.095; FeSO ₄-7H ₂O, 500; MnSO ₄-H ₂O, 154; CuSO ₄-5H ₂O, 39.2; CoCl ₂-6H ₂O, 24.8; Na ₂B ₄O ₂-10H ₂O, 17.7; Add about 0.400mL6M sulfuric acid and prevent precipitation.

The optimum condition of during fermentation operating fermentor tank comprises following parameters: 30 ℃ of pH of table 2. fermentor tank conditioning process variable set-point temperature 7.0 stir 20 liters of/minute pressure of 300RPM air-flow, 0.3 crust fermentation back step: remove silicone oil

Full nutrient solution is transferred to results jar and sedimentation 4 hours at least from fermentor tank.Usually sedimentation is spent the night.After the sedimentation, the lower layer of water that contains cis-indane two pure and mild fermentation solid moves to the pillar charging stock tank mutually.Discard top organic layer.Expanded bed adsorption

Subsequently water being pumped into density pumps greater than the bottom of 1.1 brominated styrene divinylbenzene resin column and from the top.Expanded bed operation is adsorbed onto on the resin cis-indane glycol and fermentation solid thing mistake post with being detained.Behind the application of sample, with the above flow path direction washing resin of deionized water to replace remaining fermentation solid thing and exhausted water-based feed liquid.With the upstream direction washing resin, use 20%v/v acetonitrile/80%v/v water elution liquid with the 20%v/v acetonitrile/80%v/v water elution liquid of one times of column volume then with downflow direction wash-out cis-indane glycol.The concentration of cis-indane glycol is partly collected and detected with HPLC respectively to effluent liquid.Vacuum concentration/isopropyl acetate extracting

Compile contain～the effluent liquid wash-out partial vacuum of 80% cis-indane glycol concentrates to reduce volume and to reduce ethane nitrile content.Adding isopyknic isopropyl acetate in concentrated solution also stirred 5 minutes at least.After at least 2 hours because each phase layering of gravity settling effect.Remove upper organic phase, then lower floor's water is with the extracting three times again of isopyknic isopropyl acetate.The special crystallization of vacuum concentration/chirality

This crystallisation step has two crucial purposes.A purpose is by removing other indenes metabolite, and as the indenols of carrying secretly in the initial separating step process and 2, the 3-bihydrogen-1-indenone improves chemical purity.Another purpose is by removing the unwanted 1R in the mother liquor, 2S steric isomer raising chiral purity.Crystallisation process is as follows:

The isopropyl acetate extract vacuum concentration that merges to～30g cis-indane glycol/liter, the glass filter with the mesoporosity rate filters to remove all particulate matters subsequently.Extract after the filtration further be concentrated into final concentration be 200g cis-indane glycol/liter.Cis in last concentration process-indane glycol begins crystallization, and also at least 8 hours post crystallizations of ageing are complete to be cooled to 5-10 ℃.Filter crystal,, use hexane wash subsequently with isopropyl acetate/hexane (1: 1), and vacuum-drying.From full nutrient solution to cis-typical yields of indane glycol is 45%, greater than 99.5% steric isomer excessive be 1S, the 2R stereoisomer form.Product reclaims

Product cis-(1S, 2R)-the indane glycol is present in the aqueous phase of substratum, thereby the method separation and purification of available routine, as the centrifugal of water or gravity clarification, concentrating under reduced pressure, with extractings such as conventional solvent such as isopropyl acetates, adjust pH, with conventional resin (as negatively charged ion or Zeo-karb, nonionic resin etc.) processing, with conventional sorbent material (as gac, silica gel, Mierocrystalline cellulose, aluminum oxide etc.) processing, crystallization, recrystallization etc.

The preferred sequence of method comprise water clarification, product be adsorbed onto on the resin and use solvent mixture wash-out, reduced pressure under under the solvent extraction, reduced pressure of the concentrating of rich fraction, product solvent concentrate and crystallization.Cis-(1S, 2R)-the indane glycol is to the conversion of cis-(1S)-amino-(2R)-indanol

Cis-(1S, 2R)-reaction of indane glycol and acetonitrile, and then the hydrolysis in the presence of water is finished fast.With the solid cis of monovalent-(1S, 2R)-the indane glycol is dissolved in excessive acetonitrile, adds or do not add organic solvent.Typical solvent is a methylene dichloride.Because glycol is heat release with mixing of acetonitrile, thus with to cool off usually before strong acid contacts.

Subsequently with cis-(1S, 2R)-mixture of indane glycol and acetonitrile contacts with excessive strong acid such as trifluoroacetic acid, methylsulfonic acid or sulfuric acid.Usually add about 2 normal strong acid.After about 1 or 2 hour, add excessive water.Remaining acetonitrile is removed by distillation or backflow, obtains Ritter solution.

The cis that obtains-(1S)-amino-(2R)-indanol is substantially free of trans aminoidan alcohol steric isomer.Usually, in step subsequently, do not need to split.The purifying of cis-(1S)-amino-(2R)-indanol

Ritter solution can be removed the acid pollution thing through different processing.

The A gegenion is to extracting: add alkali with neutralizing acid, and pH is risen to about 12 or higher, the Ritter solution that obtains alkalizing.The Ritter solution of this alkalization is used has any organic solvent of suitable solubleness such as methylene dichloride, ethyl acetate or 1-butanols (preferred 1-butanols) to carry out extracting to cis aminoidan alcohol.But aqueous phase discarded then.

In the organic layer that contains cis-aminoidan alcohol, add and surpass cis-normal suitable acid of aminoidan alcohol.The acid that is fit to will form salt composite, and make cis-aminoidan alcohol be easier to water-soluble solution with cis-aminoidan alcohol.Suitable acid like this includes but not limited to L-tartrate, D-tartrate, mesotartaric acid, xitix, propanedioic acid, citric acid, formic acid, HCl, preferred L-tartrate.

The salt that obtains in organic solvent is used the aqueous solution such as water extracting subsequently, obtains a water extract.Use the alkalimetric titration water extract, when the about 8-9 of pH, begin crystallization.When reaching about 11-12, finishes pH crystallization usually with alkalimetric titration.(1S) that obtains-amino-(2R)-indanol is essentially pure product.

B. cation-exchange chromatography: in addition, Ritter solution also can be removed the acid pollution thing by cation-exchange chromatography.Any Zeo-karb all is fit to, but generally includes the SDEB styrene diethylenebenzene resin, is connected to acid groups on it, as sulfonic acid or carboxylic acid.

Resin is mixed with Ritter solution, and unwanted acid is removed in the washing of water or other aqueous solvent then.Step (rising pH is the to keep cis-aminoidan alcohol solvable) wash-out that adds alkali goes out bonded cis-aminoidan alcohol, then with any wash-out in many solvents such as methyl alcohol, acetonitrile or the THF aqueous solution.Alkalization-wash-out circulation can repeat several times with wash-out cis-aminoidan alcohol from the resin quantitatively.(1S) that obtains-amino-(2R)-indanol is essentially pure product.Preparation

Can contain the unit dosage of conventional no cytotoxic drug acceptable carrier, auxiliary agent and vehicle by intermediate synthetic product compound of the present invention, by oral, parenterai administration (comprising subcutaneous injection, intravenous injection, intramuscularly, breastbone inner injection or infusion techniques), suck and spray or the administration of rectal administration mode.

Method of the present invention and intermediate are applicable to that preparation is used to suppress that hiv protease, prevention or treatment human immunodeficiency virus (HIV) infect, and the pathological condition that causes thus of treatment such as the end product compound of acquired immune deficiency syndrome (AIDS).Treatment acquired immune deficiency syndrome (AIDS) or prevention or treatment HIV infect to be defined as and include but not limited to treat HIV infection state widely: acquired immune deficiency syndrome (AIDS), ARC (AIDS related complex), no matter symptom or asymptomatic is arranged, and the contacting of actual or potential and HIV.For example, HIV after can being used for the treatment of the past and HIV has suspicious the contact by the end product compound of method of the present invention and intermediate preparation infects, for example because blood transfusion, organ transplantation, body fluid exchange, mosquito bite, unexpected acupuncture or catch patient's blood when surgical operation.

End product hiv protease inhibitor also is used to prepare and carry out the shaker test of antiviral compound, comprises with comparing.For example, the end product compound is used to separate the enzyme mutant body as the fabulous screening implement of more effective antiviral compound.And this compound is applicable to foundation or determines the binding site of other antiviral agent to hiv protease, for example passes through competitive inhibition.End product compound with method of the present invention and intermediate preparation is to be used for the Industrial products that these purposes are sold.

The end product hiv protease inhibitor compound following structure of J tool or acceptable salt of its medicine or hydrate.

Compound J called after N-(2 (R)-hydroxyl-1 (S)-indanol)-2 (R)-phenmethyl-4 (S)-hydroxyl-5-(1-(4-(3-pyridylmethyl)-2 (S)-N '-(tertiary butyl formamido group)-piperazinyl))-valeramide; (1S-(1 α (α S ^*, γ R ^*, δ (R ^*)), 2 α)-N-(2,3-dihydro-2-hydroxyl-1H-indenes-1-yl)-2-(((1, the 1-dimethyl ethyl) amino) carbonyl)-γ-hydroxyl-α-(phenmethyl)-4-(3-pyridylmethyl)-2-piperazine valeramide; Or N-(1 (S)-2,3-dihydro-2 (R)-hydroxyl-1H-indenyl)-4 (S)-hydroxyl-2 (R)-phenmethyl-5-(4-(3-pyridylmethyl)-2 (S)-tertiary butyl carbamyls)-piperazinyl) valeramide.

The hiv protease inhibitor compound of available intermediate of the present invention and method preparation is disclosed in EPO 541,168.The pharmaceutical compositions that the hiv protease inhibitor compound can contain medicine carrier and treatment this compound of significant quantity or the acceptable salt of its medicine needs the patient of this treatment.EPO 541,168 discloses the dosage of suitable pharmaceutical preparation, route of administration, salt form and this compound.

Compound of the present invention may have asymmetric center, thereby can be racemic modification, racemic mixture and discrete diastereomer or steric isomer, and all isomeric forms all comprise in the present invention.Stereoisomer mixture comprised 1: 1 mixture, and any other mixture, as 1: 4,4: 3,2: 1.

Details are as follows for the model experiment step of employing novel method.These steps are only for illustrating rather than to the restriction of novel method of the present invention.

The scheme of embodiment 1 pseudomonasputida F1

A. the anti-phase analysis of substrate and product: two parts of Virahols (IPA) are added in a full nutrient solution.With mixture shake after 1 minute with centrifugal 15 minutes of 5000rpm with sedimentation cell and from IPA/ water (upper strata) separating oil (bottom).One five equilibrium upper strata liquid is expelled to the C that links to each other with the HPLC system ₁₈The reversed-phase HPLC post is (in 250 * 4.6mm).

B. cis-1, the chiral determination of 2-indane glycol: make the full nutrient solution back (gravity settling～10 minute) that is separated, 5mL water (bottom) is added in the 10mL ethyl acetate (EthAc).The EthAc layer (upper strata) of 5mL is dry under nitrogen purging.The compound that the EthAc extracting goes out is suspended in 95% hexane subsequently again: 5% ethanolic soln.The chiral column (250 * 4.6mm) that injection links to each other with the HPLC system.

C. plant female the cultivation: C-E partly is 23 liters of fermentor tank designs in the present embodiment.500mL seed culture medium (table 1, inoculation 1mL starter culture in 2 liters of no flask with indentation above) are being housed.Seed is shaken bottle to be placed in the 200RPM rotary shaker 30 ℃ of cultivations.When culture grows to mid-log phase (about 8 hours), 50mL is moved in the production container.

D. induce: in the stirred vessel that 15 liters of production substratum (table 3) are housed, add toluene (75g).Fall link to each other with the waste pipe steam that sends with the mensuration fermentor tank of LEL meter and whether be lower than the Lower Explosive Limit (LEL) of toluene.Observed value is about 30% of toluene LEL after just having added toluene, is the high scale of whole process.Shake from the seed of 50mL C part immediately and get 50mL the bottle and inoculate into fermentor tank.All fermentor tank parameter is being induced whole process middle part with the bio-transformation stage remain unchanged (table 4) except that pH.Induce the back (about 12-14 hour) of spending the night, nutrient solution is faint yellow, can add indenes.Table 3. is used for making the substratum of indenes bio-transformation form the improvement of (Finette etc., Journal of Bacteriology 160,1004 (1984)) at the bio-reactor of 23 liters of stirrings by pseudomonasputida F1).All values is every liter.

Material	Produce the amount in the substratum
		The L-arginine	????2.00g
??(NH 4) 2SO 4	????1.00g
		??Na 2HPO 4	????5.24g
??KH 2PO 4	????2.76g
		??MgSO 4-7H 2O	????0.29g
??CaCl 2-2H 2O	????0.07g
		??(NH 4) 6Mo 7O 24-4H 2O	????0.17mg
??FeSO 4-7H 2O	????1.93mg
		The Hutners mineral substrate *	????---
Metal 44	????1.00mL
		Silicone oil	????200mL
Defoamer	????1.00mL

Transfer pH to 7.0 with 6M sulfuric acid

Table 4. fermentor tank condition

Process variable	The set-point
		Temperature	????30℃
????pH	????7.0
		Stir	????300RPM
Air-flow	5.0 rise/minute
		Pressure	0.3 crust

E. bio-transformation: indenes is directly added in the container to final concentration 1.0g/L the beginning bio-transformation.To monitor the gross product formation amount and the cis-1 of fermentation, the optically active of 2-indane glycol in 30-60 minute at interval.Cis 1,2-indane glycol concentration in about 4 hours reaches about 200mg/L of platform area.Even if from the indenes to the cis-1, the bio-transformation of 2-indane glycol stops, cis-1S, and the excessive EE of steric isomer of 2R-indane glycol also continues from being increased to greater than 98% less than 60%.Dissolved oxygen concentration in the container descends during the fermentation, and it is 40% saturated that minimum observed value reaches.8 hours bio-transformation after dates, 52% indenes is utilized, and calculates with the polarimetry purity of product to reach 39% transformation efficiency.To monitor the gross product growing amount and the cis-1 of fermentation, the optically active of 2-indane glycol in 30-60 minute at interval.8 hours bio-transformation after dates, 62% indenes is utilized, and reaches 35% transformation efficiency.Before the water layer clarification, fermentor tank is cooled to 15 ℃.

The scheme of embodiment 2F1: scale rises to 70 liters of fermentor tanks

A. plant female the cultivation: inoculation 1mL starter culture in 2 liters of no flask with indentation of 500mL seed culture medium (table 1) are housed.To shake bottle is placed in the 200RPM rotary shaker 30 ℃ of cultivations.Cultivate after 8 hours, culture reaches optical density(OD) (600nm) 0.6～0.7, can be moved to produce in the container.

B. induce: in the stirred vessel that 50 liters of production substratum (table 1) are housed, add toluene (250g).As with less fermentor tank, whether link to each other with the waste pipe steam that sends with the mensuration fermentor tank of LEL meter is lower than the LEL of toluene.Adding will begin in a minute to measure behind the toluene obtains about 30% toluene LEL once more, is the high scale in the whole process.The whole compositions that immediately seed shaken in the bottle are inoculated into fermentor tank (portion C).Except that pH, the fermentor tank parameter is being induced and two stages of bio-transformation all remain unchanged (table 2).Induce the back (about 12～14 hours) of spending the night, nutrient solution is faint yellow, can add indenes.

C. bio-transformation: directly indenes is added in the container to final concentration 2.5g/L the beginning bio-transformation.As if indenes purity (technical grade 90% or SILVER REAGENT 99+%) minimum or do not have an influence to the polarimetry purity influence of product.To monitor the gross product growing amount and the cis-1 of fermentation, the optically active of 2-indane glycol in 30-60 minute at interval.Cis-1S in about 6 hours, 2R-indane diol concentration reaches the 500-700mg/L of platform area.Cis-1S, the EE of 2R-indane glycol continues from rising to nearly 80% less than 60%.Although pH is not controlled, the pH under this under the described conditions scale never reduces to below 6.5 or rises to more than 7.5.As with 23 liters of fermentor tanks, dissolved oxygen concentration descends during the fermentation, but is not less than 40% saturated dissolved oxygen (DO).8 hours bio-transformation after dates, 62% indenes is utilized, and reaches 35% transformation efficiency.Before the water layer clarification, fermentor tank is cooled to 15 ℃.

D. water layer clarification: stop the agitator of fermentor tank, each composition of sedimentation at least 4 hours.Lower aqueous layer inserts in the bottle.The concentration of the volume of water layer and cis indane glycol is respectively 37 liters and 0.45g/L.Add deionization (DI) water (19 liters) and stirred 10 minutes in fermentor tank, sedimentation is subsequently spent the night.Secondary lower aqueous layer also inserts in the bottle.The volume of water layer and the concentration of cis indane glycol are respectively 21 liters and 0.05g/L for the second time.Silicon layer inserts in the bottle for handling.Cell in the 400mL bottle in the centrifugation water layer.Centrifugally carried out 45 minutes with 8000rpm at 5 ℃.

E. resin absorption: water layer divided and added in the post with～25mL/ clarifying first time.This post is equipped with 260mL SDEB styrene diethylenebenzene type resin (21.5 inches of the height of bed).Add the production concentration of water layer in effluent liquid and equal 25% of the production concentration that enters the mouth.In 10 a up-flow fluid, do not have detectable product, and production concentration increases to 0.1g/L rapidly in the 2 up-flow fluids of back.The total water layer that adds is 48 times of bed volumes (12.9 liters).DI washing post with 2 times of bed volumes (450mL).Contain 0.12g product (～2%) in the elutant.Use the 20% acetonitrile/80% water elution post of 3.3 times of bed volumes (900mL) subsequently, collect branch's elutriant of 0.20 times of volume (50mL).Post is with the acetonitrile of～5 times of bed volumes washing back regeneration, uses the DI water washing of several times of bed volumes again and balance again.

F. isopropyl acetate extracting: the rich fraction that in breadboard Rotary Evaporators, will merge from the 700mL vacuum concentration to 350mL.In concentrated solution, add isopyknic acetate and propyl ester and mixed 5 minutes.Gravity settling separated liquid phase at least in 30 minutes.Repeat twice of extracting again and merge in the rich fraction 94% product to reclaim.The final isopropyl acetate volume that merges is 1.05 liters.

G. carry out the special knot product of chirality with isopropyl acetate: in the Rotary Evaporators of laboratory with isopropyl acetate extract vacuum concentration to 25mL (diol concentration is about 200g/L).Concentrated solution is heated to 60 ℃ to dissolve all solids, is cooled to 55 ℃, and inoculate with 0.025g (～0.5%) two alcohol crystals.In the time more than 4 hours, this batch materials is slowly cooled to 35 ℃.Be cooled to 5 ℃ and age overnight.With sintered glass filter vacuum filtration crystal,, use the 10mL hexane wash again with the washing of 5mL cold (5 ℃) isopropyl acetate.Vacuum-drying under the case without heating.Loss of yield is about 20% in the mother liquor.

Embodiment 3 racemize cis-1, the biochemistry of 2-indane glycol splits

Pseudomonasputida F1 is with racemize cis-1, and 2-indane glycol biochemistry splits into the 1S of optically active, and the ability of 2R-indane glycol is utilized by cultivating this organism and induce to spend the night with toluene in two 23 liters of reactors.Indenes added in one of them reactor begin bio-transformation, when the bio-transformation in this reactor stops (about 3 hours), make two in the reactor cell precipitation and be resuspended in the reactor that does not add indenes in the exhausted substratum, change over to again and added 200mg/L racemize cis-1, in 2 liters of no flask with indentation of 2-indane glycol.The exhausted substratum and the aseptic seed substratum that are obtained by the culture that does not add indenes add 20% oil in contrast.All bottles that shake all are placed in the reciprocating type shaking table (per minute shakes for 200 times) 30 ℃ of cultivations.Interval measurement EE was until 26 hours (table 5).Sample also with the reversed-phase HPLC analysis to detect unwanted cis-1, any possible metabolite that generates in the apparent biochemical conversion of 2-indane glycol steric isomer.Cultivate after 26 hours, the EE that shaking of cell measure in the bottle is housed is 1S greater than 98%, the 2R steric isomer, and no matter whether added indenes in the culture.And still be racemic cis-1 in the bottle acellular shaking, 2-indane glycol.

Table 5. splits research summary

Condition	Cell is arranged		Acellular
					Indenes (+)	Indenes (-)	The substratum of no indenes	Aseptic seed substratum+oil
	T=0 hour	Racemize	Racemize	Racemize					Racemize
T=6 hour					ee＝48％	??ee＝＞98％	Racemize	Racemize
	T=26 hour	ee＝＞98％	??ee＝＞98％	Racemize					Racemize

This fractionation acts in 23 liters of scales and further confirms, obtaining final EE when repeating the step of front is 93%.In this batch, also obtained required 1S, the fabulous EE (98%) of 2R-indane glycol, and proved the repeatability of present method.

The extensive scheme fermentation of embodiment 4F1:

A. seed domestication (seed tram): used organism is pseudomonasputida F1 (wild-type).Adopt the domestication of single step seed.Seed culture medium contains 30g/l tryptone beans peptone substratum and need not to transfer pH.The freeze-drying pipe of culture thaws in room temperature, gets the 0.5ml amount and is used for inoculating 2.8 liters of 500ml seed culture mediums that shake bottle." cultivated 8 hours in 30 ℃ with 220rpm, 2 by the amplitude of oscillation on rotary shaker for seed.Contain and two shake bottle (obtaining the seed cumulative volume is 1 liter), and be used for inoculation and go into to produce fermentor tank.

B. production phase:, in three 200 gallons of fermentor tanks, add deionized water to 630 liters of working volumes for four experiments each.Directly in each jar, add medium component (seeing Table 6).Transfer pH to 7.0 with sulfuric acid or sodium hydroxide.Substratum was 121-123 ℃ of sterilization 35 minutes.

Table 6 is produced substratum and is contained following ingredients (every liter): composition farming degree Sodium phosphate dibasic 10.5g potassium primary phosphate 5.5g arginine 4.0g ammonium sulfate 2.0g magnesium sulfate heptahydrate 0.6gCaCl ₂2H ₂O 130mgZnSO ₄7H ₂O 22mgFeSO ₄7H ₂O 14mgEDTA 5mgMnSO ₄H ₂O 3mgCuSO ₄5H ₂O 0.8mgCoCl ₂6H ₂O 0.5mg (NH ₄) ₂MoO ₄4H ₂O 0.36mgNa ₂B ₄O ₇10H ₂O 0.35mg defoamer 1mg silicone oil 100g

C. fermentation condition: fermentation is carried out at 30 ℃ earlier, back-pressure 0.5 crust, air-flow 100slpm, stirring velocity 100rpm.During the fermentation back-pressure, air-flow, and stirring velocity raises automatically, and dissolved oxygen intensity is in or surpass 50% of the saturation value of an atmosphere air in substratum to keep.With 25% sulfuric acid and 50% sodium hydroxide solution pH is controlled at 6.8～7.4.

D. induce and bio-transformation: in the time of 4-8 hour, add in the fermention medium with Chemap pin, synthetic rubber tube and a peristaltic pump solution with 3.25kg toluene in 20 liters of silicone oil and 1.63kg indenes.

E. gather in the crops: monitor the coefficient of oxygen utilization (OUR) in every batch fermentation.After about 24 hours, OUR is brought down below 1mmol/l/h.6-10 hour results were a collection of after OUR descended.The degraded (cause steric isomer is excessive to be risen to greater than 97% from about 60%) of unwanted isomer takes place during this period.Every is 330mg/l than mean value.

The scheme I. of F1 tunning separation A. removes silicone oil by the gravity settling effect among the embodiment 5 front embodiment

Fermentation culture is gathered in the crops to 1500 gallons stainless steel vessel.Full nutrient solution sedimentation is spent the night.The lower aqueous layer that will contain product and fermentation cell is packed in two 200 gallon cans.Pack in 55 gallons the bucket for handling in useless silicon layer and middle layer.B. expanded bed adsorption

Prepare one to be equipped with～glass column (12 inch diameters * 96 inchages) of 85 liters of brominated styrene divinylbenzene resins.The water-based nutrient solution with 1.5～2.5gpm flow velocity from column bottom injection port sample introduction.Exhausted nutrient solution effluent liquid from the capital end shift out and be collected in 55 gallons the bucket.If in the exhausted feed liquid, detect less than product, then it pumped in the chemical soil pipe with HPLC.If in the exhausted feed liquid, detect product then keep barrel content and when next batch begins, be adsorbed onto on the post again.When all water-based nutrient solutions all go up sample in the post after, upwards wash post with deionized water, from post, remove residual nutrient solution and solid.Washing is upwards washed post with 95% water/5% acetonitrile of 100 gallons later.Contain water-acetonitrile and the washing fluid be collected in 55 gallons the bucket in.The spawn that contains when next batch begins in water-acetonitrile and the washing fluid all heavily is adsorbed onto on the resin.Stop to flow and make resin sedimentation again.Remove liquid above the resin with pump from the capital end.Product is with 100 gallon of 20% acetonitrile/80% water elution, and effluent liquid is collected in 5 gallon bottles.Resin is with 50 gallons of acetonitrile regenerated from washing and with the deionized water wash of several times of bed volumes balance again.C. rich fraction concentrates/the isopropyl acetate extracting

The rich fraction of product vacuum concentration in 50 gallons of glass lined reactor.Rich fraction continuous sample introduction makes amount of liquid maintain the maximum functional volume until adding all rich fractions.Being concentrated into final volume is 25 gallons.This process helps to reduce to foam in the aqueous concentrates and acetonitrile minimum.Utilize residual vacuum that isopyknic isopropyl acetate is added in the water layer.Contents stirred 5 minutes, gravity settling is stratified liquid after at least 1 hour.The residual water of lower floor is packed in 55 GPB, and the organic extract in upper strata is also packed in one 55 GPB.Any milkiness liquid layer is always along with water layer shifts out together.Water layer reinstalls in 50 gal reactor, with the extracting three times again of isopyknic isopropyl acetate, reclaims the product more than 95% in the rich fraction that merges.Poor extract (3 and 4) remains the first extracting that is used for next batch to reduce solvent load.D. isopropyl acetate concentrates/crystallization

In 50 gallons of glass lined reactor, isopropyl acetate extract partial vacuum is concentrated into the 7-10 gallon.Enriched material moves in 5 gallon bottles.Subsequently the line filter (line filter) of enriched material by a mesoporosity degree added in one 20 liters of Rotary Evaporators, remove some brown particulate matters.Extract further be concentrated into about 200 the gram cis-indane glycol/liter.Cis in last concentration process-indane glycol begins crystallization, is cooled to 5-10 ℃ and age overnight post crystallization and finishes.Vacuum filtration crystal and with isopropyl acetate, isopropyl acetate/hexane (1: 1) and the hexane wash crystal of cold (5 ℃) on a sintered glass filter.Vacuum-drying crystal under heating condition not.

The scheme kind of embodiment 6421-5 is female grows: 2 liters of no flask with indentation that the 300ml seed culture medium is housed are used to prepare to be used for the inoculum of 23 liters of production containers.Seed culture medium is made up of 3% trypticase soy broth (TSB).Seed culture medium is autoclaving 20 minutes on the spot.The freeze-drying pipe that the 421-5 culture is housed is thawed, and shake the culture aseptic condition that after bottle is cooled to room temperature 300 μ l is thawed at seed and be displaced downwardly to seed and shake in the bottle.Shake bottle and cultivated 14 hours with 200-250RPM in shaking table at 30 ℃, the entire contents of shaking in the bottle with seed is inoculated 23 liters of fermentor tanks then.Fermentation/bio-transformation (23 liters of scales): the substratum that the chemistry that table 8 is listed is determined adds in 23 liters of production containers, and working volume is 15 liters.Listed the process variable of fermentation in the table 7.

Table 7

Process variable	First set-point	Other condition
			Temperature	????30℃	All the time control
??pH	????7.20	All the time control
			Back-pressure	0.34 crust	All the time control
Stir	????450	Change is to control dissolved oxygen more than 50%
			Air-flow	????7.51pm	Change is to control dissolved oxygen more than 45%
Dissolved oxygen	????100％	With stirring gentle current control more than 45%

All salt and the soybean oil of adding were also sterilized 1 hour on the spot in 23 liters of fermentor tanks.Glucose is sterilized separately and is once added in the batch when beginning.Inoculate this batch with whole 300ml inoculums, and under the listed process variable condition of table 7, be cultured to 28 hours.

Table 8 is used for the composition that substratum is determined in half of pseudomonasputida 421-5 fermentation

Constituent concentration

1. glucose ^#20mM

2.(NH ₄) ₂SO ₄?????????????????10g/l

3.K ₂HPO ₄??????????????????????6g/l

4.FeSO ₄·7H ₂O?????????????????0.03g/l

5?MgSO ₄·7H ₂O?????????????????1.2g/l

6.EDTA ^*?????????????????????????0.0167g/l

7. soybean oil 20% (v/v)

8.A-9 solution 90ml

The # initial concentration, the speed with control adds glucose thereafter.

^*Just add when having only with glucose.(SILVER REAGENT, 99+%), the next one after 10 hours per hour replenished 0.3g/l in 10 hours in batches, per 2 hours additional in batches 0.6g/l in following 8 hours to add indenes with 2.5g/l during beginning.Begin with NaOH pH to be controlled at 7.2, (about 10 hours) used NH afterwards ₄OH control is to avoid nitrogen hunger.Ferment after 6 hours, glucose adds continuously with 4g/L/hr.Stop after adding 6-8 hour continuously adding.In the whole process of this batch, with cascade system dissolved oxygen is controlled at more than 45% with stirring and air-flow.Air-flow control beginning after stirring velocity reaches maximum value 750RPM.Use this scheme, ferment and reach the peak value 2g/L (80%EE) of indane glycol after 20 hours.

The separation of embodiment 7 tod mutant

A. cultural method: pseudomonasputida F1 (DSM 6899) and mutant thereof are cultivated and are kept with tryptone beans peptone agar (TSA) or trypticase soy broth (TSB).All cultures are 30 ℃ of cultivations.Plate culture is in 4 ℃ of storage 2-4 weeks, as the inoculum source of transformation experiment.Liquid culture is stored with the dilution in 1: 1 of 20% (v/v) glycerine and in-70 ℃ of branches such as branch.In culture medium prescription, mend 20g/L agar and prepare solid medium.In order to cultivate the solid medium culture in the presence of toluene vapor, the 3-5ml toluene of 100 * 15mm glass culture dish in the 15ml glass test tube that will contain the solid medium of selection is placed in the glass moisture eliminator, and cultivates under suitable temperature.

B. indenes is to cis-1S, the conversion of 2R-indane glycol-shake a bottle scale:

Contain the basic salt culture medium (MMC) of Citrate trianion or not the basic salt culture medium (MM) of carbonaceous sources be used for indenes to cis-1S, the conversion of 2R-indane glycol, its (g/l) composed as follows: 3-(N-morpholino)-propane sulfonic acid (MOPS), 20.9; Sodium Citrate, usp, Dihydrate Powder, 2.94; K ₂HPO ₄, 2; (NH ₄) ₂SO ₄, 1; MgSO ₄7H ₂O, 0.4; FeSO ₄7H ₂O, 0.010; A9 solution, 2.5ml; PH, 7.2.A9 solution composition following (mg/L): H ₃BO ₃, 300; ZnCl ₂, 50; MnCl ₂4H ₂O, 30; CoCl ₂, 200; CuCl ₂2H ₂O, 10; NiCl ₂6H ₂O, 20; NaMoO ₄2H ₂O, 30.Mend 20g/l agar in the superincumbent prescription and prepare solid medium.The basic salt culture medium of 20ml adds the 5ml soybean oil or silicone oil is sterilized in 250ml three baffle plate Erlenmeyer flasks.After sterilization and the cooling, in bottle, add indenes or toluene on demand.

Cell after washing grows the 25ml overnight culture when being used for transformation experiment on TSB, centrifugal subsequently and be resuspended in the 20ml MM substratum.With 2.5ml silicone oil and 1g/l indenes (by whole culture volume), cell suspension is moved in a 250ml three baffle plate Erlenmeyer flasks.Culture is in 30 ℃, and 180RPM rotation stir culture 24 hours is taken out a culture supernatant (water-based) afterwards and analyzed cis-1S with reversed-phase HPLC, the concentration of 2R-indane glycol.

In addition, indenes is to cis-1S, and the conversion of 2R-indane glycol is undertaken by the metabolism cell that enlivens in the shake-flask culture thing.The one 250ml three baffle plate Erlenmeyer flasks that 20ml MMC substratum, 5ml soybean oil and 2.5g/l indenes (by whole culture volume) be housed are with the 0.2ml TSB culture inoculation of spending the night.Production is shaken bottle and is as above cultivated, each shake bottle suitably extracting to determine cis-1S, 2R-indane glycol amount and steric isomer purity.For time course experiment, each several bottles that shake of cultivating are all inoculated, and extracting is all shaken bottle to determine cis-1S, the amount of 2R-indane glycol and steric isomer purity on each time point.

The separation method of the culture that C. non-toluene relies on: two kinds of sudden changes and screening method are used for being created in substratum does not need toluene indenes can be changed into cis-1S, the pseudomonasputida F1 mutant of 2R-indane glycol.The core of two kinds of screening methods is that many microorganisms that utilize aromatic hydrocarbons are converted into indoles ability (Ensley etc., Science, 222,167 (1983) of indigo (blue pigments); Clarke P.H. etc., FEMS Microbiol.Lett.24,109 (1984)).Tod changes into cis-indoles-2 with indoles, 3-dihydrodiol (Clarke etc., 1984).The spontaneous removal of water forms indolol, that oxidation by air subsequently produces is indigo (Ensley etc., above, nineteen eighty-three).

Cultivation became mazarine at the pseudomonasputida F1 bacterium colony on the toluene vapor in 18-24 hour, just acquisition is blue and cultivation (contains the non-carbon source of inducing) in substratum under the normal air condition bacterium colony need reach 36 hours.Therefore indoles can be used as the active useful biochemical marker of dioxygenase to indigo conversion, and detects the dioxygenase activity when being used in toluene and not existing.

In method 1, cell dilutes 10 in 30 ℃ of overnight incubation and with phosphoric acid buffer (100mM, pH 7.2) in TSB ^-4Contain 3ml MMC substratum add 0 (contrast), 10,20,30,40,50,75 or some 18 * 150mm test tubes of 100mg/l ICR 191 with the inoculation of 0.1ml dilution inoculum.Tube culture in the dark 30 ℃ with the 220RPM stir culture.Cultivate after 16-18 hour, 30mg/l ICR 191 cultures only have trace growth, and 20mg/l ICR 191 cultures have half level of growth of the contrast of being similar to.ICR 191 concentration surpass 30mg/l and suppress growth fully, and 10mg/l concentration does not have and can detectedly influence growth.Centrifugal results 20mg/l ICR 191 cells in culture are used the phosphoric acid buffer washed twice.

The separation of D mutant strain 42l-5: carry out viable count in the pipe that selects after, the diluent (not merging thereby observe bacterium colony) that 1ml is fit to is applied to mineral substance substratum (the 0.040g/l FeSO that contains improvement ₄) and contain the 1mM indoles as the culture dish of dioxygenase activated indicators (among the 24.5cm * 24.5cm).After about 36 hours, several flat pannel display go out the existence of several mazarine bacterium colonies in 30 ℃ of cultivations.Choose and repave on the identical substratum 16 bacterium colonies altogether are aseptic from the flat board.After cultivating 36 hours again, each of these strain isolateds all is inoculated in the pipe that the 5ml trypticase soy broth is housed.After 18 hours, add aseptic silicone oil of 1ml and 5 μ l indenes in 30 ℃ of cultivations in each pipe.Each pipe was cultivated 24 hours under identical culture condition again.After centrifugal, detect the existence of indane glycol in the supernatant liquor with reversed-phase HPLC.Under this non-inductive condition, find that strain isolated 421-5 produces about 426mg/l indenes, and its parent's culture is 100mg/L.Mutant 421-5 (a.k.a.MB 5652) is preceding isolating, and we have checked about 200,000 bacterium colonies.

For method 2, a TSB overnight culture of centrifugal results is also used citrate buffer solution (100mM, pH 5.5) washed twice.Add N-methyl-N '-nitro-N-nitrosoguanidine (NTG) to final concentration 50mg/l, and in room temperature stir culture every now and then.Cultivate after 30 minutes, cell also suitably dilutes with the phosphoric acid buffer washed twice.

The sudden change group (0.1ml) of some equal portions is applied on the solid indoles indicator substratum in 1.00 * 15mm glass culture dish, and in the presence of toluene in 30 ℃ of cultivations.Choose white colony (not having indigo generation, dioxygenase and toluene feminine gender) to TSA.The negative bacterium colony of single dioxygenase is inoculated into to be equipped with contains silicone oil and do not contain mutually in the 250ml three baffle plate Erlenmeyer flasks of minimum medium of Citrate trianion as second.In the culture of silicone oil in mutually, add toluene to whole total concn 5g/l.30 ℃ with 180RPM rotation wave and culture 48-72 hour in, the positive reverse mutation strain of spontaneous toluene begins to grow.Separate the bacterium colony of these reverse mutation strain cultures, and be determined in the shake flask fermentation process indenes, the conversion that the non-toluene of 2R-indane glycol relies on to cis-1S.Isolating in this way such culture is 419-11R3.

Embodiment 8A. cis-1S, 2R-indane glycol is to the transformation experiment of cis-1S-amino-2R-indanol

Material	Amount	Molecular weight	Mole number
				Cis-1S, 2R-indane glycol	100g	?150.18	???0.66
20% oleum (sulfuric acid/sulphur trioxide)	66.2mL	??98.08	???1.33
				Acetonitrile	633.3mL
Water	1000mL

With cis-1S, 2R-indane glycol (100g, 0.66 mole) is cooled to-25～-30 ℃ then in 25 ℃ of adding acetonitriles (633mL).The oleum solution (66.2mL, 1.33 moles) of adding 20% keeps temperature to be lower than-10 ℃.Make mixture be warming up to 20 ℃ after all adding, aging 1.5 hours, add entry (1000mL) then.The distillation acetonitrile solvent reaches nearly 100 ℃ until internal temperature, and mixture was worn out 4.5 hours under this temperature.With solution concentration to 100 gram aminoidan alcohol/liter.Productive rate 86.7% is greater than 99%ee.B. begin the extracting aftertreatment to be separated (-) cis-ammonia from glycol with the gegenion of Ritter solution

The base indanol

Material	Amount	Molecular weight	Mole number
				The steps A product	(548.7g 50.0g cis-aminoidan alcohol)	??149.2	??0.335
The 1-butanols	501mL
				50％NaOH	145mL	???40
Water	625mL
				L-tartrate	60.0g	??150.1	??0.4

In 2 liters of round-bottomed flasks that thermometer and overhead are housed, add 548.7g Ritter solution (cis of 50.0g steps A-aminoidan alcohol) and 167ml 1-butanols.Begin to add 50%NaOH, keep temperature to be lower than 40 ℃ with water-bath simultaneously.Continue to add and be higher than 12 until pH.Added 103ml 50%NaOH altogether.Mixture darkens in adding the NaOH process.

Mixture is placed the separating funnel layering.Use 2 * 167mL 1-butanols extracting water layer then.Merge three organic layers and use 60.0g L-tartrate (1.2 molar equivalent) the solution extracting that is dissolved in the 250mL water.Organic layer is further used the extracting of 3 * 125mL water after the layering.The water layer that merges then vacuum concentration to 220mL.Some solids have begun precipitation.

Enriched material is drenched one with 30mL water to be equipped with in the 500mL round-bottomed flask of thermometer and overhead.Begin to add 50%NaOH.In adition process, with water-bath with temperature maintenance below 45 ℃.In 8～9 cis of pH-aminoidan alcohol beginning crystallization.Continue to add NaOH to pH and be higher than 12.Added 42mL 50%NaOH altogether.Mixture is slowly cooled to 0-5 ℃, aging 2 hours, filter (slowly filtering) also with 0～5 ℃ of water washing of 150ml.In vacuum drying oven, blow 45 ℃ of dryings 18 hours with nitrogen.The output of (-) cis-aminoidan alcohol: 45.74g (96.6wt%); 2.1% trans-aminoidan alcohol.C. beginning with Dowex 50 * 8 resin columns Ritter solution to be carried out aftertreatment from glycol separates

(-)-cis-aminoidan alcohol experiment

Material	Amount	Molecular weight	Mole number
				The steps A product	550mL (50.0g cis-aminoidan alcohol)	?149.2	?0.335
Dowex 50 * 8 (100 order) resin	500mL (in the water)
				50％NaOH	34.5g	???40
Water	600
				The 20%MeCN aqueous solution	2175

Prepare a 500mL Dowex 50 * 8 (100 order) resin column.Water is just being washed earlier back backwash post.With 1.5 times of bed volumes/hour flow velocity glycol Ritter solution (the 50.0g cis-aminoidan alcohol in the 550mL steps A product) is gone up sample in post.Wash post with 600mL then.The rate of leaking is less than 1%.

At first, try with NaCl wash-out but efficient is not high.Reclaimed 12.3g cis-aminoidan alcohol altogether.Use following NaOH wash-out again after washing post with the 500mL 20%MeCN aqueous solution.

Dissolving 20.3g 50%NaOH in the 250mL 20%MeCN aqueous solution.This solution is upwards pumped in the post.Use 100mL 20%MeCN drip washing then.Placed 1.75 hours.Beginning is with 1.5-2 times of bed volume flow velocity wash-out post.When solvent arrives resin bed, with 750mL 20%MeCN aqueous solution wash-out.The cis aminoidan alcohol total amount that wash-out goes out is 16.7g.

Dissolving 11.5g 50%NaOH in the 250mL 20%MeCN aqueous solution.This solution is upwards pumped in the post.Use the continuous drip washing of 100mL 20%MeCN then.Placed 1.75 hours.Beginning is with 1.5-2 times of bed volume flow velocity wash-out post.When solvent arrives resin bed, with 750mL 20%MeCN aqueous solution wash-out.The cis that wash-out goes out-aminoidan alcohol total amount is 16.7g.

Dissolving 2.7g 50%NaOH in the 250mL 20%MeCN aqueous solution.This solution is pumped in the post.Beginning is with 1.5-2 times of bed volume flow velocity wash-out post.When solvent arrives resin bed, with 500mL 20%MeCN aqueous solution wash-out.The cis that wash-out goes out-aminoidan alcohol total amount is 3.0g.

With dense HCl the pH of the merging elutriant of the first two times wash-out is transferred to 2.95.Then with this solution for vacuum concentration to 228mL.This solution is used for different crystallization experiments subsequently.

Overall recovery with the cis-aminoidan alcohol of NaCl and NaOH wash-out is 48.7g.

The preparation of embodiment 9 acid amides 9

With an anhydrous THF of 17.8L (KF=55mg/mL) (KF represents Karl Fisher water titration(method)) and a triethylamine (868mL who is equipped with in the 50L round-bottomed flask of thermocouple probe, mechanical stirrer and nitrogen inlet junctor and bubbler, 6.22mol) in (-)-cis-1-aminoidan-2-alcohol (884g, 5.93mol) solution, be cooled to 15 ℃.(1000g 5.93mol), remains on internal temperature between 14-24 ℃ with frozen water cooling bath simultaneously to add 3-phenyl propionyl chloride in the clock time at 75 minutes then.After adding, mixture wore out 30 minutes between 18-20 ℃, with the disappearance of HPLC analytical review (-)-cis-1-aminoidan-2-alcohol.

Reaction process is analyzed monitoring with high performance liquid chromatography (HPLC): 25cm Dupont C ₈-RX post, 60: 40 acetonitrile/10mM (KH ₂PO ₄/ K ₂HPO ₄), the 1.0mL/ branch, last sample volume=20mL detects wavelength=200nm, sample=500 times diluent.Approximate retention time:

Retention time (minute) the signing result

6.3 cis-aminoidan alcohol

10 minutes (pH of the mixture behind the usefulness equal-volume water dilution 1mL sample is between 4.3-4.6) handled and stirred to reaction solution usefulness tosic acid pyridinium salt (241g, 0.96mol, 0.16 equivalent).Then, add 2-methoxyl group propylene (1.27L, 13.24mol, 2.2 equivalents) and with reaction solution 38-40 ℃ of heating 2 hours.Reaction mixture is cooled to 20 ℃ also with ethyl acetate (12L) and 5%NaHCO ₃The aqueous solution (10L) distributes.Stir the mixture and make it layering.Use 5%NaHCO ₃The aqueous solution (10L) and water (4L) washing ethyl acetate extract.The ethyl acetate extract utilizes the air distillation drying and changes solvent into hexanaphthene (the about 30L of cumulative volume).When distillation and concentrated the end (the ethyl acetate extract of 20% volume percent), make hot cyclohexane solution slowly cool to 25 ℃ of crystallizations and go out product.The slurries of gained further are cooled to 10 ℃ and aging 1 hour.The filtering separation product is also with cold (10 ℃) hexanaphthene (2 * 800mL) washing wet cakes.Filter cake after the washing obtains 1.65kg acetonide 9 (86.4%, the HPLC98% peak area) in 40 ℃ of vacuum (26 " Hg) drying. ¹H NMR (300.13MHz, CDCl ₃, main rotational isomer) δ 7.36-7.14 (m, 9H), 5.03 (d, J=4.4,1H), 4.66 (m, 1H), 3.15 (m, 2H), 3.06 (wide s, 2H), 2.97 (m, 2H), 1.62 (s, 3H), 1.37 (s, 3H); ¹³C NMR (75.5MHz, CDCl ₃, main rotational isomer) and δ _C168.8,140.9,140.8,140.6,128.6,128.5,128.4,127.1,126.3,125.8,124.1,96.5,78.6,65.9,38.4,36.2,31.9,26.5,24.1.Ultimate analysis calculated value C ₂₁H ₂₃NO ₂:

C，78.47；H，7.21；N，4.36。Measured value: C, 78.65; H, 7.24; N, 4.40.

The tosylate method of embodiment 10 epoxide 11 preparations

Acetone solvate 9 (1000g among the 15.6L THF (KF=22mg/ml) in one 50L, the four neck round-bottomed flasks of thermopair, mechanical stirrer, addition funnel and nitrogen inlet junctor are housed, 3.11mol) and 2 (S)-toluenesulphonic acids glycidyl esters, 10 (853g, 3.74mol, 1.2 equivalent) solution purges by vacuum nitrogen and bleeds three times, and is cooled to-56 ℃.In 2 hour time, added hexamethyl two silicon lithium nitride (LiN[(CH then ₃) ₃Si] ₂) (2.6L, 1.38M, 1.15 equivalents), keep internal temperature simultaneously between-50～45 ℃.Reaction mixture was warming up to-25 ℃ then in-45～-40 ℃ of stirrings 1 hour in 1 hour time.Mixture stirs 4 hours (or be 3.0% peak area until initial acetone solvate) between-25～22 ℃.

Reaction process is analyzed monitoring with HPLC: 25cm * 4.6nm Zorbax silicagel column, and the hexane solution of 20% ethyl acetate, the 2.0mL/ branch, applied sample amount=20mL detects wavelength=254nm, sample=100 times diluent.Approximate retention time:

Retention time (minute) qualification result

5.5 acid amides 9

6.5 toluenesulphonic acids glycidyl ester 10

13.5 epoxide 11

Reaction mixture distributes with-15 ℃ deionized water (6.7L) quenching and with ethyl acetate (10L).Stir the mixture and make it layering.Use 1%NaHCO ₃The mixed solution washing ethyl acetate extract of the aqueous solution (5L) and saturated NaCl (0.5L).Ethyl acetate extract (28.3L) concentrates with vacuum distilling (28 " Hg) and adds ethyl acetate to whole solvents in addition and changes ethyl acetate (final volume=11.7L) into.The ethyl acetate enriched material further change solvent be MeOH with crystallized product, and be concentrated into final volume 3.2L.Add 10L methyl alcohol and collect the 10L distillate to remove residual ethyl acetate solvent.The slurries that obtain are cooled to 5 ℃ and aging 0.5 hour then in 22 ℃ of stirrings 1 hour.The filtering separation product is also used cold methanol (2 * 250mL) washing wet cakes.Filter cake after the washing in 25 ℃ of vacuum (26 " Hg) drying obtain 727g epoxide 11 (61.2%, HPLC analyze account for main epoxide area 98 7%). ¹³C?NMR(300MHz，CDCl ₃)171.1，140.6，140.5，139.6，129.6，128.8，128.2，127.2，126.8，125.6，124.1，96.8，79.2，65.8，50.0，48.0，44.8，39.2，37.4，36.2，26.6，24.1。

The preparation of embodiment 11 penult compounds 14

Be equipped with Virahol (2-propyl alcohol in one 72 liter of four neck round-bottomed flask of mechanical stirrer, reflux exchanger, steam bath, teflon-coated thermopair and nitrogen inlet, 18.6L) in 2 (S)-tertiary butyl carboxylic acid amides-4-N-Boc-piperazine 12 (1950g, 6.83mol,＞99.5%ee) (the ee=steric isomer is excessive) and epoxide 11 (2456g, 97.5: 25 4S.R epoxide mixture, slurries reflux 6.51mol) (internal temperature is 84-85 ℃).After 40 minutes, obtain uniform solution.Mixture heating up refluxed 28 hours.

Internal temperature is 84-85 ℃ in reflux course.Reaction process is analyzed monitoring with HPLC: 25cm Dupont C ₈-RX post, 60: 40 acetonitrile/10mM (KH ₂PO ₄/ K ₂HPO ₄), the 1.0mL/ branch detects wavelength=220nm, applied sample amount=2 μ l, reaction mixture with dilution in acetonitrile to 1mL.Approximate retention time is:

Retention time (minute) qualification result

4.8 piperazine 12

8.9 epoxide 11

15.2 coupled product 13

After 28 hours, remaining epoxide 11 and coupled product 13 (HPLC analysis) are respectively 1.5% peak area and 91-93% peak area.Mixture is cooled to 0-5 ℃ and add 20.9L 6N HCl, and temperature remains on below 15 ℃ in this process.After adding HCl mixture is warming up to 22 ℃.Observed gas and emitted (iso-butylene) this moment.Mixture was worn out 6 hours at 20-22 ℃.

Reaction process is analyzed monitoring with HPLC: condition is the same.Approximate retention time is:

Retention time (minute) qualification result

7.0 cis-aminoidan alcohol

11.9 penult compound 14

15.1 coupled product 13

Mixture is cooled to the 0 ℃ of also slow 7.5L of adding 50%NaOH mixture pH is transferred to pH=11.6.Temperature remains on below 25 ℃ in this process.Mixture distributes with ethyl acetate (40L) and water (3L).Stir the mixture and make it layering.Organic phase (60L) is reduced pressure (29 " Hg) concentrate and solvent is changed to DMF and is concentrated into final volume 10.5L (KF=.1.8mg/mL).14 productive rate is 86.5% in the HPLC mensuration ethyl acetate.Penult compound 14 among the DMF is directly used in next step experiment without being further purified.For isolating 14: ¹³C NMR (75.4MHz, CDCl ₃) δ 175.2,170.5,140.8,140.5,139.9,129.1,128.5,127.9,126.8,126.5,125.2,124.2,73.0,66.0,64.8,62.2,57.5,49.5,47.9,46.4,45.3,39.6,39.3,38.2,28.9.

The preparation of embodiment 12 compound J monohydrates

Compound J

Be dissolved in the back DMF (10.5L, KF=10mg/mL) 14 solution add the DMF that the 8L sieve does (KF＜30mg/L), and with mixture " steam bath heating under the Hg vacuum, the main steaming dewaters and/or Virahol or ethyl acetate solvent that all are remaining 30.Final enriched material volume is 13.5L (KF=1.8mg/mL), in 25 ℃ solution, add then triethylamine (2.86L, 20.51mol), add again 3-picoline chloride hydrochloride (96%, 1287g, 7.84mol).The gained slurries are heated to 68 ℃.

Reaction process is analyzed monitoring with HPLC, and condition ditto goes on foot.Approximate retention time is:

Retention time (minute) qualification result

2.7?????????????????DMF

4.2 the 3-methyl is than pyridine muriate

4.8 compound J

9.1 penult compound 14

Mixture is analyzed in 68 ℃ of aging penult compound 14 usefulness HPLC until remnants and is shown less than 0.3% peak area.

Mixture is cooled to 25 ℃ also with ethyl acetate (80L) and the saturated NaHCO of 24L then in 68 ℃ of stirrings 4 hours ₃The mixed solution of the aqueous solution and distilled water (14L) distributes.Mixture stirs and makes it layering in 55 ℃.Ethyl acetate layer is in 55 ℃ of waters (20L) washing three times.The ethyl acetate layer of washing is concentrated into final volume 30L under barometric point.Under atmospheric pressure concentrate when finishing, in hot solution, add entry (560mL) and mixture is cooled to 55 ℃.Inoculate with compound J monohydrate.Mixture is cooled to 4 ℃, filters and collect product.(2 * 3L) washed product, and dry in 25 ℃ of vacuum (-tight) housings obtain 2905g (70 7%) compound J monohydrate white solid with cold ethyl acetate.

Embodiment 13 pyrazines-2-tertiary butyl carboxylic acid amides 17

2-pyrazine carboxylic acid (8) 3.35kg (27mol) oxalyl chloride 3.46kg (27.2mol) TERTIARY BUTYL AMINE (KF=460 μ g/ml) 9.36L (89mol) EtOAc (KF=56 μ g/ml) 27LDMF 120mL1-propyl alcohol 30L

Carboxylic acid 16 is suspended at N ₂Descend among the 27L EtOAc and 120mL DMF in the churned mechanically 72L three-necked flask, and suspension is cooled to 2 ℃.Add oxalyl chloride, simultaneously temperature is remained between 5～8 ℃.

In 5 hours, add oxalyl chloride.In the adition process of heat release, emit CO and CO ₂The HCl major part that forms is stayed in the solution.The precipitation that occurs may be the HCl salt of pyrazine acyl chlorides.By carrying out the mensuration that acyl chlorides forms with the dry-out sample in the TERTIARY BUTYL AMINE quenching reaction.Reaction finishes back remaining sour 16 less than 0.7%.

It is very important that acyl chlorides forms the mensuration of finishing, because incomplete reaction will cause the formation of dual-tert-butyl oxamide impurity.

This reacts available HPLC monitoring: 25cm Dupont Zorbax RXC8 post, and 1mL/ divides flow velocity, and the detection wavelength is 250nm; Use 98%0.1%H in the time of 30 minutes ₃PO ₄The aqueous solution and 2%CH ₃CN is to 50%H ₃PO ₄The aqueous solution and 50%CH ₃The linear gradient elution of CN.Retention time: sour 16=10.7 branch, acid amides 17=28.1 branch.

Reaction mixture wore out 14 o'clock in 5 ℃.The gained slurries are cooled to 0 ℃ and add TERTIARY BUTYL AMINE, and adding speed will keep internal temperature to be lower than 20 ℃.

Adding needs 6 hours, because reaction is very heat release.The hydrochloric acid uncle fourth ammonium that sub-fraction produces is separated out from reaction solution, is fluffy white solid.

Mixture was worn out 30 minutes in 18 ℃ again.Sedimentary ammonium salt is shifted out in filtration.Filter cake washs with 12LEtOAc.The organic phase 6L 3%NaHCO that merges ₃With the saturated NaCl solution washing of 2 * 2L.Organic phase is filtered with 200g Darco G60 activated carbon treatment and with Solka Folk.Filter cake washs with 4L EtOAc.

Activated carbon treatment has been removed some purples in the product effectively.

17 EtOAc solution is concentrated into 25% of original volume in 10 millibars.Add 30L 1-propyl alcohol, continuing to be distilled to final volume is 20L.

At this moment, EtOAc is lower than ¹The detectability of H NMR (＜1%).Internal temperature is lower than 30 ℃ in this solvent changes.The backflow under barometric point of 1-propyl alcohol/EtOAc solution of 3 was stable in the time of several days.

Obtain a tawny solid, fusing point 87-88 ℃ behind a sample evaporation. ¹³C?NMR(75MHz，CDCl ₃，ppm)161.8，146.8，145.0，143.8，142.1，51.0，28.5。

Embodiment 14 racemize 2-tertiary butyl carboxylic acid amides piperazines 18 Material

2.4kg (13.4mol) the 1-propanol solution 12L20%Pd (OH) of pyrazine 2-tertiary butyl carboxylic acid amides 17 ₂/ C 16% (weight) water 144g.

The 1-propanol solution of pyrazine-2-tertiary butyl carboxylic acid amides 17 is put into 5 gallon autoclave.Add catalyzer, mixture is at 40psi (3 normal atmosphere) H ₂In in 65 ℃ of hydrogenations.

24 hours afterreactions have absorbed the hydrogen of theoretical amount, and GC (gas-chromatography) shows 17 less than 1%.Cooling mixture is also used N ₂Purge, filter with Solka Floc and shift out catalyzer.Catalyzer washs with 2L temperature 1-propyl alcohol.

Discovery has improved filter effect and has reduced the loss of product in the filter cake with warm 1-propyl alcohol when washing leaching cake.

Reaction is monitored with GC: 30m Megabore post, be warming up to 160 ℃ with 10 ℃ of/minute speed from 100 ℃, and kept 5 minutes, be warming up to 250 ℃, retention time with 10 ℃/minute then: 17=7.0 branch, 18=9.4 branch.React also available TLC (thin-layer chromatography) monitoring, as solvent, triketohydrindene hydrate is as developping agent with EtOAc/MeOH (50: 50).

Show behind the sample evaporate to dryness that amidation and hydrogenant overall yield are that 88%, 18 concentration is 133g/L.

Obtain behind the sample evaporate to dryness 18 for white solid, fusing point is 150-151 ℃; ¹³C NMR (75MHz, D ₂O, ppm) 173.5,59.8,52.0,48.7,45.0,44.8,28.7.

Two (the S)-camsilates (S)-19 of embodiment 15 (S)-2-tertiary butyl carboxylic acid amides piperazine Material racemize-2-tertiary butyl carboxylic acid amides piperazine 18 4.10kg (22.12mol)

1-propanol solution 25.5kg solvent (S)-(+)-10-camphorsulfonic acid 10.0kg (43.2mol) 1-propyl alcohol 12L acetonitrile 39L water 2.4L

The 1-propanol solution adding one of amine 18 is connected with in the 100L flask of batch concentration device.Solution is lower than 25 ℃ in 10 millibars is concentrated into about 12L.

At this moment product is precipitated out from solution, but product is dissolved in the solution again when mixture heating up to 50 ℃.

The homogeneous sample analysis shows that 18 concentration is 341g/l.Concentration is measured by HPLC: 25cmDupont Zorbax RXC8 post, and flow velocity 1.5mL/ branch, the detection wavelength is 210nm, (98/2) CH ₃CN/0.1%H ₃PO ₄Aqueous solution isocratic elution.18 retention time: 2.5 minutes.

Obtain clarifying little brown solution after adding acetonitrile (39L) and water (2.4L).

The water-content of KF titration determination and ¹The CH that H NMR integration is measured ₃CN/1-propyl alcohol ratio shows CH ₃CN/l-propyl alcohol/H ₂The O ratio is 26/8/1.6.Concentration in the solution is 72.2g/L.

With 30 fens clock times in 20 ℃ of branches, 4 batches of addings (S)-10-camphorsulfonic acids (CSA).Temperature rises to 40 ℃ after adding CSA.Form thick white precipitate behind the several minutes.White slurries are heated to 76 ℃ with the dissolving all solids, make little brown solution then in 8 hours internal cooling to 21 ℃.

Product is in 62 ℃ of precipitations.Product 21 ℃ of aging i.e. filtrations, is not used 5L CH ₃CN/l-propyl alcohol/H ₂O 26/8/1.6 solvent mixture washing leaching cake.It is used N in 35 ℃ of vacuum ovens ₂Purge drying, get 5.6kg (39%) 19, be white crystalline solid, fusing point 288-290 ℃ (decomposition), [α] _D ²⁵=18.9 ° of (c=0.37, H ₂O). ¹³C?NMR(75MHz，D ₂O，ppm)222.0，164.0，59.3，54.9，53.3，49.0，48.1，43.6，43.5，43.1，40.6，40.4，28.5，27.2，25.4，19.9，19.8。

Detect according to following chirality HPLC, the ee in the material is 95%: a 19 samples (33mg) are suspended in 4mL EtOH and 1mL Et ₃Among the N.Add Boc ₂O (11mg) made reaction mixture aging 1 hour.Remove whole solvents in vacuum, resistates is dissolved among about 1mLEtOAc.Contain SiO through one ₂Pasteur's dropper filter, make eluent with EtOAc, each product fraction of evaporate to dryness is dissolved in hexane, about 1mg/ml again.Separation of stereoisomers on Daicel Chiracell AS post with hexane/IPA (97: 3) solvent system, flow velocity 1mL/ branch, detects wavelength 228nm.Retention time: S enantiomorph=7.4 minute, R=9.7 branch.

Embodiment 16 is by salt 19 preparation (S)-2-tertiary butyl carboxylic acid amides-4-tert-butoxycarbonyl-piperazines 12

Two (S)-(+) of material (S)-2-tertiary butyl carboxylic acid amides piperazine-CSA salt 19,95%ee 5.54kg (8.53mol) two dimethyl dicarbonate butyl ester 1.86kg (8.53mol) Et ₃N 5.95L (42.6mol) EtOH Punctilious 200 proof 55LEtOAc 2L

In (the S)-CSA salt 19 that has in the 100L three-necked flask of application of sample funnel in N ₂Under add EtOH, add triethylamines in 25 ℃ then.Adding Et ₃Solid is easy to dissolving behind the N.With Boc ₂O is dissolved in EtOAc and is added in the application of sample funnel.Add Boc ₂The speed of O EtOAc solution remains on below 25 ℃ temperature.Application of sample continues 3 hours.Add Boc ₂Made reaction mixture behind the O solution aging 1 hour.

React available HPLC monitoring: 25cm Dupont Zorbax RXC8 post, flow velocity 1mL/ branch, the detection wavelength is 228nm; Transfer (50/50) CH of pH=6.8 with NaOH ₃CN/0.1M KH ₂PO ₄Isocratic elution.12 retention time=7.2 minute.Carry out chiral determination with the system identical with back.React also available TLC monitoring, the EtOAc with 100% is as solvent (R _f=0.7).

In the batch-type thickener of one 10 millibars of vacuum, be lower than under 20 ℃ of conditions solution concentration to about 10L at internal temperature.Slowly adding 20L EtOAc lays equal stress on and is concentrated into about 10L and finishes solvent switch.With 60L EtOAc reaction mixture is washed in the extractor.Use 16L 5%Na ₂CO ₃The aqueous solution, 2 * 10L deionized water and 2 * 6L saturated sodium-chloride water solution washing organic phase.Carry the water washes of merging and wash organic phase with 20L EtOAc back suction with 2 * 3L water and 2 * 4L saturated sodium-chloride water solution.The EtOAc extract that merges is in 10 millibars of vacuum, and internal temperature is lower than in 20 ℃ the 100L batch-type thickener and is concentrated into about 8L.Slowly adding about 20L hexanaphthene lays equal stress on and is concentrated into about 8L and makes solvent change hexanaphthene into.In slurries, add 5L hexanaphthene and 280mL EtOAc, reflux when all reagent all dissolve post-heating.Cooling solution also adds crystal seeds (10g) in 58 ℃.In 4 hours, slurries are cooled to 22 ℃, at 22 ℃ of aging 1 hour after-filtration separated products.With 1.8L hexanaphthene washing leaching cake, and in vacuum oven 35 ℃ of N ₂Purge drying, obtain 1.87kg 12 (77%, the HPLC analytical results is greater than 99.9% peak area, and the R-isomer is lower than detection level), be little tawny powder.[α] _D ²⁵=22.0 ° (c=0.20, MeOH), 107 ℃ of fusing points; ¹³C NMR (75MHz, CD ₃Cl ₃, ppm) 170.1,154.5,79.8,58.7,50.6,46.6,43.6,43.4,28.6,28.3.

The preparation of embodiment 17 racemic indene oxides

With indenes (95%, 122mL) be dissolved in methyl alcohol (812mL) and acetonitrile (348mL), filter then.Filtrate transfers to pH 10.5 with the 1M aqueous sodium hydroxide solution then with 0.05M bibasic sodium phosphate (116mL) dilution.(35%, 105mL) water (53mL) dilution, and added with 3 hour time keeps temperature to be in 25 ℃ to aqueous hydrogen peroxide solution in this process, and is 10.5 with 1M aqueous sodium hydroxide solution (being total to 120mL) maintenance internal pH.

After 6 hours, add the 1M sodium metabisulfite aqueous solution (26mL), add the 1M NaOH aqueous solution (39mL) in this process and keep pH to be higher than 8.3.Add entry (700mL) and use methylene dichloride (580mL and 300mL) extracting mixture.Organic extract that will contain the merging of indene oxide (117g) is concentrated into 600mL.

The hiv protease of embodiment 18 microbial expressions suppresses to measure

The inhibition research of the proteolytic enzyme of escherichia coli expression and peptide substrates (Val-Ser-Gln-Asn-(β naphthyl) Ala-Pro-Ile-Val is 0.5mg/mL during the reaction beginning) reaction was carried out 1 hour in 30 ℃ among the pH 5.5 at the 50mM sodium acetate.The inhibitor that will be dissolved in the different concns of 1.0 μ lDMSO adds in the 25 μ l peptide aqueous solution.Add the 0.33nM proteolytic enzyme (0.11ng) that 15 μ l are dissolved in 0.133M sodium acetate pH 5.5 and 1% bovine serum albumin solution and start reaction.Reaction stops with 160 μ l, 5% phosphoric acid.Reaction product is separated (the wide hole of VYDAC 5cm C-18 reversed-phase column, acetonitrile gradient, 0.1% phosphoric acid) with HPLC.The inhibition degree of reaction is determined by the peak height of product.The HPLC of independent synthetic product is as quantitative criterion and the proof formed as product.The IC of compound J ₅₀Be about 0.6nM.

Although the specification sheets of front has been introduced principle of the present invention, also provide and be intended to illustrational embodiment, be to be understood that enforcement of the present invention comprises all general changes, correction and improvement, they are all within below claims and equivalent scope thereof.

Claims (13)

1. One kind synthetic (1S, 2R)-method of indane glycol, this method may further comprise the steps:

   A) a certain amount of tod is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) obtain (1S, 2R)-the indane glycol.
2. One kind synthetic be substantially free of any stereoisomer (1S, 2R)-method of indane glycol, this method may further comprise the steps:

   A) a certain amount of tod is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) purifying (1S, 2R)-the indane glycol;

   D) the step c) product is carried out the special crystallization of chirality;

   E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol.
3. One kind synthetic (1S, 2R)-method of indane glycol, this method may further comprise the steps:

   A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) obtain (1S, 2R)-the indane glycol.
4. 4 one kinds synthetic be substantially free of any stereoisomer (1S, 2R)-method of indane glycol, this method may further comprise the steps:

   A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) purifying (1S, 2R)-the indane glycol;

   D) product to step c) carries out the special crystallization of chirality;

   E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol.
5. 5. the method for synthetic (1S) who is substantially free of any stereoisomer-amino-(2R)-indanol, this method may further comprise the steps:

   A) a certain amount of tod is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) purifying (1S, 2R)-the indane glycol;

   D) product to step c) carries out the special crystallization of chirality;

   E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol;

   F) with the step e) of monovalent (1S, 2R)-the indane glycol is dissolved in and obtains second kind of mixture in the excessive acetonitrile, second kind of mixture is maintained at about between-40 ℃～25 ℃; G) to wherein sneaking into excessive normal strong acid, and reaction is maintained at about-40 ℃～25 ℃ between;

   H) obtain being substantially free of (1S) of any stereoisomer-amino-(2R)-indanol.
6. 6. the method for synthetic (1S) who is substantially free of any stereoisomer-amino-(2R)-indanol, this method may further comprise the steps:

   A) a certain amount of dioxygenase is contacted with a certain amount of indenes;

   B) fermentation gained mixture;

   C) purifying (1S, 2R)-the indane glycol;

   D) product to step c) carries out the special crystallization of chirality;

   E) obtain being substantially free of any stereoisomer (1S, 2R)-the indane glycol;

   F) with the step e) of monovalent (1S, 2R)-the indane glycol is dissolved in and obtains second kind of mixture in the excessive acetonitrile, second kind of mixture maintained approximately between-40 ℃～25 ℃;

   G), and reaction maintained approximately between-40 ℃～25 ℃ to wherein sneaking into excessive normal strong acid;

   H) obtain being substantially free of (1S) of any stereoisomer-amino-(2R)-indanol.
7. One kind from cis-(1S, 2R)-indane two pure and mild cis-(1R, 2S)-remove in the mixture of indane glycol cis-(1R, 2S)-the biological method for splitting of indane glycol, this method may further comprise the steps:

   A) make a certain amount of dihydrodiol desaturase and cis-(1S, 2R)-indane two pure and mild cis-(1R, 2S)-the mixture contact of indane glycol;

   B) fermentation gained mixture;

   C) obtain being substantially free of the steric isomer cis-(IR, 2S)-cis of indane glycol-(1S, 2R)-the indane glycol.
8. 8. pseudomonasputida 421-5 is the pure growth of ATCC 55687.
9. 9. claim 5 or 6 method, this method further comprises following additional step:

   I) to step h) product carry out gegenion to extracting;

   J) obtain pure basically (1S)-amino-(2R) indanol.
10. 10. claim 5 or 6 method, this method further comprises following additional step:

    I) to step h) product carry out cation-exchange chromatography;

    J) obtain pure basically (1S)-amino-(2R) indanol.
11. 11. each method in the claim 1,2 or 5, wherein tod also claims ATCC 55688 from pseudomonasputida F1.
12. 12. each method in the claim 3,4 or 6, wherein dioxygenase also claims ATCC 55687 from pseudomonasputida 421-5.
13. 13. the method for claim 7, wherein the dihydrodiol desaturase also claims ATCC 55688 from pseudomonasputida F1, or pseudomonasputida 421-5, also claims ATCC 55687.