CN1163612A - Preparation of immunogens and other conjugates of drugs - Google Patents

Abstract

The invention provides a reactive derivative of dialkyl amino compounds, particularly dialkyl amino drugs, for facilitating the conjugation of the drug, directly or through a bifunctional spacer, to a carrier compound such as proteinaceous materials. The derivative has formula (a) wherein D is the drug and n is an integer greater than 1, and preferably equal to 2. The drug derivative carrier conjugate can be used as an immunogen for production of antibodies specific to the drug. Additionally, the conjugate can be coupled to a solid support, such as a polymer particle, for use as a particle reagent in immunoassays specific to the drug.

Description

The preparation of immunogen and other medicines binding substances

Invention field

The present invention relates to bridged piperazine derivatives and have the immunogen of dialkyl amino group and the application in other the drug conjugates in preparation.

Background of invention is described

Accept usually necessary its serum of periodic monitoring of individuality of pharmacological agent or the levels of drugs in other body fluid.The method that detects the some drugs level has been arranged.For example, the serum level of lignocaine (lidocaine, a kind of antiarrhythmics) can be used gas-chromatography, high performance liquid chromatography or zymetology immune analysis determination.

The scattering of light immunoassay provides a kind of highly sensitive immunoassay means, and it uses the particle reagents (particle reagent) with high refractive index, for example at United States Patent (USP) 4,401,765 and 4,480, the particle described in 042 strengthens than turbid inhibition immunoassay.Automatic Clinical Analyzer, the aca of Du Pont Automatic Clinical Analyzer for example, single dose reagent and patient's sample mix in automatically will packaged in advance detection packing, with sample and reagent at required temperature (37 ℃), insulation required time, reading and print result then.Such automatic analyser is because its result's accuracy, repeatability and use have easily become in the clinical labororatory requisite.Immunoassay and some enzyme immunoassays of scattering of light immunoassay, alloimmunization analysis, affinity column mediation can be finished on automatic analyser.Must be ready to the reactive derivatives bonded particle reagents of goal analysis thing or some other solid support and the specific antibody of analyte in these sensitive detect, to use.

So far, the lignocaine particle reagents that also is not suitable for described automatic analyser.Gas phase and highly effective liquid phase chromatography detection method are unsuitable for using in automatic analyser.

Lignocaine comprises a dialkyl amino group in its structure,

It is believed that many medicines that other has dialkyl amino group yet are not suitable for the particle reagents that automatic analyser uses.

Pronestyl is the another kind of medicine that has dialkyl amino group in its structure,

In a kind of known reagent, protein binding is indicating ^*The methyl site; In another kind of reagent, protein binding indicates in following formula ^*The site.

All contain dialkyl amido in the structure of imipramine and amitriptyline (amitriptyline), the method for developing its antibody has been described in the document.In two kinds of methods, the structure of compound is all passed through additional isolation arm on the aromatic nucleus of compound (spacer arm) and is modified.Then, the isolation arm is linked on the bovine serum albumin and is used to produce antibody.Referring to Adamczyk, M. etc., immunological method magazine, 162 (1) volumes, 47-58 page or leaf (1993) and 163 (2) volumes, 187-97 page or leaf (1993).

People need a kind of dialkyl amido medicine that promote to be attached on the albumen so that the method for immunogen and particle reagents to be provided, and this immunogen and particle reagents are used to sensitiveer, rapid and reliable automatic analysis technology.Need be used to prepare the reactive derivatives of immunogen and antigenic dialkyl amido medicine especially, they are applicable in the commercial automatic analyser that is used for lignocaine and other dialkyl amido medicine in the detection by quantitative human serum.

Summary of the invention

The present invention relates to a kind of component that is used in conjunction with carrier compound, wherein this component has following formula:

Wherein n is the integer more than or equal to 1, and D is a kind of compound, and preferably a kind of medicine has dialkyl amino group in its non-derived structure.In the preparation of component of the present invention, the dialkyl amino group of this compound is replaced by following residue,

This residue is a kind of derivative that is similar to piperazine, and it has kept the structure of Compound D basically, has added the reactive terminal nitrogen-atoms on the carrier compound for being attached to simultaneously.

Randomly, a difunctional spacer can be incorporated on the terminal nitrogen atom so that reactive derivatives component of the present invention is attached on the carrier compound.Difunctional spacer can be selected from cyclic anhydride, two N succinimide radical derivative or dialdehyde.

Carrier compound can be (I) proteinaceous substances, for example lockhole limpet hemocyanin, ovalbumin or bovine serum albumin (BSA), or (ii) synthetic polymeric material, for example polyethylenepolyamine or polyoxyethylene glycol.Deutero-component-carrier compound binding substances-can be used as the immunogen that produces the Compound D specific antibody, perhaps can be attached on the solid support (for example polymer beads), with reagent as the level of detection compound D in immunoassay by carrier compound.

A kind of preferred particle reagents of the present invention comprises a kind of polymer beads with kernel and shell, and wherein kernel is when the sodium D-line wavelength measurement, has the polymkeric substance that is not less than 1.54 specific refractory power.Shell is following polymer of monomers: (i) double-stranded unsaturated monomer, its have can with the functional group with the reaction of the relevant nucleophilic compound of biology; The (ii) double-stranded unsaturated monomer of Ren Xuan other, its content is enough to produce the insoluble polymer particle, and the interior vouching aggressiveness that (iii) is no more than shell 10 weight parts, shell be with the covalently bound kernel of compositions derived therefrom (above-mentioned carrier compound binding substances) in the presence of form by polyreaction.

Accompanying drawing is described

Accompanying drawing shows the success of the inventive method, its shows be by per minute the milli absorbancy at 340nm place with sample in the figure of the agglutination rate measured of the variation (mA/min) of lignocaine concentration.

Detailed description of the preferred embodiments

The invention provides a kind of promotion compound and (specifically, be the medicine with dialkyl amido Thing), by replacing dialkyl amino group with piperazine, the new method of being combined with carrier compound. Carry The body compound can be protein material, for example antigen or enzyme. By be derivatized to comprise piperazine structure and The additional amino group that provides has promoted cohesive process. If need, can be at the terminal ammonia of piperazine Insert sept between base group and the carrier.

The invention provides a kind of reactive derivative with dialkyl amido medicine of following structure:

Wherein D is any medicine, in the structure that it does not develop, usually contains dialkyl amino group, N is equal to or greater than 1 integer, preferably between 1 to 3, most preferably equals 2. The number of the n of every dialkyl group chain of reactive derivatives of the present invention equates. Not combination of albumen To the dialkyl group chain, and combination is not by the effect length of chain, so the length of chain is for piperazine derivatives The function of thing or bond is also indecisive. And, in original medicine with the length of dialkyl group chain Change over the piperazines structure and can not change antibody to the identification of medicine.

Alternatively, this component can comprise a difunctional sept to link reactive medicine bridged piperazine derivatives and protein carrier compound, and its structure is as follows:

Wherein n ' is from 0 to 6 integer. Also can use other difunctional sept. For example, Difunctional sept such as cyclic anhydride, two N succinimide radical derivatives or dialdehyde for amino group can To be used as difunctional sept. Can use many other possible difunctional septs. For example, ginseng See in " protein combination and cross-linking chemistry " (CRC publishing house 1991) of S.S.Wong The same difunctional sept (homobifunctional spacer) that 4 chapter 76-97 page tables 1 are listed, and the The isodigeranyl function sept (heterobifunctional spacer) that 5 chapter 152-167 page tables 1 are listed.

For with reactive medicaments derivative as antigen, for instance, a kind of protein is connected on the terminal nitrogen atom or in the functional group of difunctional sept, and is as follows:

or

Alternatively, medicaments derivative-the protein conjugate that is with or without spacer can be used to medicine is fixed on the solid support, this solid support for example is described in United States Patent (USP) 4,401, particle reagents in 765 and 4,480,042, this patent is incorporated herein by reference, or any suitable solid support surface.Medicaments derivative-carrier conjugates of the present invention is particularly suitable for the immunoassay that carries out on above-mentioned conventional Automatic Clinical Analyzer.There is the different albumen of many kinds can be used for association reaction.The final application of medicine-protein conjugate is depended in its selection.For example, if binding substances be used to medicine attached on the solid support or on the particle with as the antigen in the immunoassay, can use a kind of connector (linker), as polyethylenepolyamine (PEPA) and human serum protein (HSA) or bovine serum albumin (BSA).Identical binding substances can be used as immunogen.But, owing to immunogenicity and specific reason, preferably different protein carriers.If binding substances will be used as immunogen, preferably BSA, ovalbumin, lockhole limpet hemocyanin (KLH).Different protein carriers is attached on the reactive medicine bridged piperazine derivatives with known technology, as S.S.Wong described in " protein binding and cross-linking chemistry) " (CRC press 1991).

The individual example of the dialkyl amido medicine that can from bridged piperazine derivatives of the present invention, the be benefited I that is listed in the table below.

Table I

Medicine

The structure of the medicine of name structure piperazine derivatives

Lignocaine (toponarcosis; Anti-heart disorder)

Pronestyl (anti-heart disorder)

Noxiptiline (antidepressant)

Nortriptyline (antidepressant)

When as immunogen, reactive medicaments derivative of the present invention produces a kind of antibody, and this antibody is than having higher specificity with the issuable antibody of prior art for essential drugs (basic drug).Because the structure of piperazine and the structure of dialkyl amino group are very approximate, so bridged piperazine derivatives has kept the essential drugs structure.

Unique not being both of gained compound and essential drugs added an amino, this amino effectively cyclisation be present in dialkyl amido side chain in the essential drugs structure.Prior art is a site coupling protein in medicines structure, has changed its basic structure like this.

The amino of piperazine is used to medicine is coupled on the albumen to generate immunogen with known technology.(referring to S.S.Wong, quoted passage is the same) so, the structure of medicine has been retained, and do not take place significantly to change, and the site at dirt settling place does not harm medicine.

Be the preparation of bridged piperazine derivatives of explanation dialkyl amido medicine of the present invention and the embodiment of application below.

Following derived from lidocaine thing has been synthesized and has been characterized: N-lignocaine and N-lignocaine propionic acid.

Be used for immunogen and particle reagents synthetic derived from lidocaine thing 1. lignocaines

2. lignocaine propionic acid

The immunogen that preparation is following: N-lignocaine-BSA, N-lignocaine-KLH, N-lignocaine propionic acid-BSA, N-lignocaine propionic acid-KLH.Known KLH is an immunogen preferably, but its low-solubility makes it be difficult to operation and characterizes.Therefore, also prepared the BSA binding substances to back up.These immunogens can be used to open the beginning with existing technology production lignocaine antibody.

It is crucial correctly selecting a kind of reactive derived from lidocaine thing for the preparation of immunogen and particle reagents, otherwise this immunogen just can not produce the lignocaine specific antibody, and particle reagents can not be specifically combines with lignocaine among the patients serum.

The lignocaine particle reagents is to prepare from N-lignocaine-HSA, N-lignocaine-PEPA1300 (polyethylenepolyamine, molecular-weight average 1300), N-lignocaine propionic acid-HSA and N-lignocaine propionic acid-PEPA200.When using available from the anti-lignocaine antibody test of the breadboard goat of Kallestad, all particle reagents have all shown very high activity on the Cary19 spectrophotometer.Use contains the phosphoric acid salt (pH7.8) of 0.15M of 2.5% polyoxyethylene glycol 800 (PEG), 0.1% sodium lauryl sulphate (SDS) as analysis buffer, and the particle reagents of 12 μ l and the anti-lignocaine antibody of 8 μ l are incubated together.When particle reagents during, both observed anti-HSA activity and also observed nonreactive HSA activity with the anti-lignocaine antibody test of several the rabbits.

The lignocaine particle of having drawn N-lignocaine-HSA-particle reagents strengthens than turbid inhibition immunoassay curve, the results are shown in the accompanying drawing.The agglutination rate variation is by the variation of drawing 340nm milli absorbancy per minute the curve of lignocaine concentration in the sample to be measured.The existence of sample Chinese traditional medicine has suppressed aggegation, so drug level is high more, agglutination rate is low more.This analysis is not optimized, but has reached good separation in therapeutic domain.This lignocaine particle reagents of presentation of results and immunogen are suitable for the lignocaine particle and strengthen than turbid inhibition immunoassay.And the therapeutic domain of lignocaine (1.0-12 μ g/ μ l) is well within particle strengthens than turbid inhibition immunoassay sensitivity range (about 0.5 μ g/ml-50 μ g/ml).

EXAMPLE Example 1: derived from lidocaine thing synthetic

The preparation of a.N-lignocaine

1.N-chloracetyl-2, the 6-xylidene(s).24mL 2 in the 160mL Glacial acetic acid, 6-xylidene(s) solution (0.2 mole) is cooled to 10 ℃ in ice bath, and the chloroacetyl chloride of 17.1mL (0.22 mole) once all adds.Vigorous stirring mixed solution 10 minutes once adds half saturated sodium acetate soln 200mL.Generate white precipitate immediately.The mixed solution water complements to 500mL, at room temperature stirs 30 minutes, stirs 1 hour at 4 ℃ then.Filter and collect white powder, recrystallization in methanol aqueous solution obtains white tiny needle-like crystal, and fusing point 145-146 ℃, dry weight 32.5 grams (83%).

2.N-the preparation of lignocaine.Piperazine sample (17.2g, 0.2 mole) heating is dissolved in the ethyl acetate of 150mL.In hot solution, add the N-chloracetyl-2 that b.1 3.98g be dissolved in the 50mL ethyl acetate makes from above-mentioned steps, 6-two-Tolylamine (0.02 mole), mixing solutions refluxed 30 minutes.Cooling was filtered this solution to remove the piperazine chloride of generation after 30 minutes in ice bath.Filtrate with the water washing of 20mL three times, and is used anhydrous sodium sulfate drying.70 ℃ were removed and to desolvate in 1 hour in rotatory evaporator.The oiliness resistates solidifies during cooling.With its collection and dry under vacuum condition.Dried throw out has following character: fusing point 109-114 ℃, weight 3.82g (77%), NMR (deuterochloroform): ppm start from the low field orientation of tetramethylsilane; δ 2.2 (6H, singlet), δ 2.6 (4H, triplet), δ 2.9 (4H, triplet), δ 3.1 (2H, singlet) and δ 7.0 (3H, singlet).

The preparation of b.N-lignocaine-propionic acid.B.2 the N-lignocaine (5 moles) of the 1.23g that obtains from above-mentioned steps and the mixed solution of iodopropionic acid (6.25 mmole) the 20mL acetonitrile of 1.25g were in 70 ℃ of heating 1 hour.In mixed solution, add the triethylamine of 1.25mL, continue heating 30 minutes.Mixed solution cooled off in ice bath 30 minutes.Precipitation is collected with filtration method, with cold acetonitrile washing and dry.The exsiccant precipitation has following character: fusing point 250-252 ℃, weigh 1.5 grams (94%), and NMR (deuterium is for trifluoroacetic acid): ppm starts from the low field orientation of tetramethylsilane; δ 2.2 (6H, singlet), δ 3.2 (2H, triplet), δ 3.85 (2H, triplet), δ 4.2 (8H, wide singlet), δ 4.7 (2H, singlet) and δ 7.1 (3H, singlet).Embodiment 2: lignocaine binding substances synthetic

A.N-lignocaine-protein conjugates

1. ethyl-3-(3-the dimethylaminopropyl)-carbodiimide hydrochloride (EDPC) that in 300mg HSA (purifying from the level V branch, i.e. fraction V) is dissolved in the solution of 17mL water, adds 300mg.The pH value transfers to 6 (by adding the hydrochloric acid of 0.1M), and adding is dissolved in 3mL alcoholic acid 150mg N-lignocaine.The pH value transfers to 6 again.Mixed solution at room temperature stirred 90 minutes, added the EDPC of other 150mg then.Mixed solution spends the night 4 ℃ of stirrings, uses the thoroughly dialysis of phosphoric acid buffer (pH value 7.8) of 15mM then.

2. preparation N-lignocaine-BSA and N-lignocaine-KLH binding substances use the same method.

The preparation of b.N-lignocaine-propionic acid binding substances

1. N-lignocaine-propionic acid of 160mg (0.5 mmole) solution (using molecular sieve drying) of being dissolved in the 10mL dimethyl formamide cools off in ice bath, the triethylamine (0.5 mmole) that adds 139 μ L adds the isobutyl chloroformate (0.5 mmole) of 130 μ L afterwards.Mixed solution stirred 20 minutes at 4 ℃, added to comprise the water of 300mg PEPA-200 to 15mL.Mixed solution was preserved 30 minutes at 4 ℃, at room temperature preserved then 5 hours.Need not dialyse and to use this product.

2. except that phosphoric acid buffer (the pH value 7.8) dialysis of product with 15mM, other step is identical with preparation PEPA-200 binding substances.Embodiment 3: the preparation of lignocaine particle reagents (PR)

N-lignocaine-HSA of a.N-lignocaine-HSA-PR 1mg/mL, 0.4% particulate material and 0.18% GAFAC are dissolved in the solution of 34mL 15mM phosphoric acid buffer (pH value 7.5) 70 ℃ of heating 45 minutes.Mixed solution cools off in ice bath, usefulness Sorvall RC-5B whizzer centrifugal 90 minutes in 19k rpm.Abandoning supernatant, precipitation is resuspended in the phosphoric acid buffer of 15mL 15mM.The particle reagents recentrifuge, precipitation is resuspended in the glycine (pH value 9.0) of 5mL 15mM and 0.01% thimerosal (thimerosal) with supersound process.

B.N-lignocaine-PEPA-PR, N-lignocaine-propionic acid-HSA-PR and N-lignocaine-propionic acid-PEPA-PR use identical method to prepare other lignocaine particle reagents except that the pH value that is used for synthetic PEPA particle reagents is 9.5.Embodiment 4: particle strengthens the reactivity than turbid inhibition immunoassay (PETINIA)

The reactivity of the PETINIA of lignocaine particle reagents is measured in the 1mL 150mM phosphoric acid buffer that comprises 2.5%PEG and 0.1%SDS, the anti-lignocaine antibody of 8 μ L, 10 μ L demarcation agent and 12 μ L particle reagents.Under room temperature, follow the tracks of speed of reaction at 340nm with the Cary19 spectrophotometer.

When with the anti-lignocaine TPPA of rabbit, several particle reagents have provided about 400mA/ minute speed, but reaction can not be suppressed by lignocaine.When with the anti-lignocaine TPPA of goat, obtain identical speed, and 80% can be suppressed by lignocaine.The results are shown in Table II.

Table II

The used caliberator speed of the anti-lignocaine antibody of reactive lignocaine particle reagents (PR) of lignocaine particle reagents

(μ g/mL) (mA/min) N-lidocaine-HSA-PR rabbit 0 600N-lidocaine-HSA-PR rabbit 12 540N-lidocaine propionic acid-PEPA-PR rabbit 0 400N-lidocaine propionic acid-PEPA-PR rabbit 12 380N-lidocaine-PEPA-PR rabbit 0 360N-lidocaine-PEPA-PR rabbit 12 330N-lidocaine-HSA-PR goat 0 510N-lidocaine-HSA-PR goat 1 350N-lidocaine-HSA-PR goat 5 250N-lidocaine-HSA-PR goat 12 175N-lidocaine-HSA-PR goat 20 125N-lidocaine propionic acid-PEPA-PR goat 0 360N-lidocaine propionic acid-PEPA-PR goat 1 270N-lidocaine-PEPA-PR goat 0 370N-lidocaine-PEPA-PR goat is the development sequence of N-acetyl procainamide (NAPA) or procainamide below 1 270.

Generate in the bridged piperazine derivatives process of pronestyl and NAPA, a committed step is to generate two (amino-ethyl) piperazine (BAP).Shown in above-mentioned reaction sequence I, the reaction of piperazine and chloromethyl cyanide to be providing two (cyano methyl) piperazine (BCP), and reduces BCP to generate two (amino-ethyl) piperazine (BAP) by the pressurized catalysis hydrogenation.BAP and NHS-ester (II)

Coupling forms acetyl pronestyl bridged piperazine derivatives (APP, NAPA bridged piperazine derivatives).Introduce the protein conjugates connexon by the reaction of APP and succinyl oxide, it and albumen coupling are to provide NAPA protein conjugates then.Synthetic the comparing with NAPA of pronestyl protein conjugates also comprises a protection and de-protected step.Shown in following reaction sequence II; uncle's fourth oxygen formamyl (t-BOC) group is used to protect the amino group of p-aminobenzoic acid to generate uncle's fourth oxygen carbamyl phenylformic acid (BOC-BA); BOC-BA and two succinimdyl carbonates (DSC) react in tetrahydrofuran (THF) (THF) and triethylamine (TEA) solution to generate NHS-ester II ', uncle's fourth oxygen carbamyl succinimide benzoic ether (BOC-SB) then.

After pronestyl and protein binding, shown in reaction sequence III, the BOC blocking group is removed with trifluoroacetic acid in organic solution (methylene dichloride).

Use the above-mentioned BAP approach of deriving, synthesized two NAPA and two pronestyl protein conjugates (having ovalbumin and KLH).The limitation of this approach is the high-pressure hydrogenation reaction of BCP, and it causes the BAP productive rate lower, about 15-20%.

The synthetic embodiment 5:BCP of NAPA bridged piperazine derivatives and protein conjugates thereof and BAP's is synthetic

21.535g piperazine (0.25 mole), 77mL triethylamine (TEA) (0.55 mole) and 300mL methylene dichloride are put into the flask of a 1000mL.34.81mL chloromethyl cyanide (0.55 mole) mixes with the 100mL-methylene dichloride, stirs afterwards to add piperazine solution.Reaction solution obtains a large amount of solid precipitations stirring at room 16 hours.Add 100mL HCl (1N) solution to extract product (reaction solution extracts three times at least, 100mL * 3).Twice of 50mL washed with dichloromethane of the HCl aqueous solution.The pH value of solution is adjusted to 11-12 with sodium hydroxide solution (3N).Solid collected by filtration, wash with water then and in a vacuum drying obtain 20g BCP, productive rate about 50%.Determine the structure of compound with nucleus magnetic resonance (NMR), the result is as follows: ¹H NMR (400MHz, CDCl ₃) δ ppm2.67 (8H, s), 3.55 (4H, s).

Inject 20g-two (cyanogen methyl) piperazine (BCP) (0.122 mole) in the bomb (high-pressure bomb, high pressure resistant metal vessel commonly used); Add then 2-3g be suspended from the 25mL95% alcoholic acid draw in (Raney) nickel catalyzator.Add 25mL ethanol again with cleaning catalyst.Close body, introduce the liquid ammoniacal liquor of about 15g (0.882 mole) from steel cylinder.Insert the hydrogen of tank pressure (15001b) then, and be warming up to 90 ℃.When hydrogen no longer is absorbed after 2-3 hour, closes well heater and make the body cooling.Bleed off hydrogen and ammoniacal liquor, play intravital composition and wash out with two parts of 50mL 95% ethanol.Remove by filter catalyzer, remove with the rotary evaporation method then and desolvate.The brown oil of gained further use vacuum distilling (70-80 ℃/5mmHg) purifying to be to obtain about 4.2g BAP, productive rate about 21%.The overall yield that obtains BAP from piperazine is about 10%.NMR result is as follows: ¹H NMR (400MHz, DMSO-d ₆) δ ppm1.87 (2H, s), 2.23-2.25 (2H, m), 2.37 (2H, m), 2.58-2.71 (6H, m), 3.66 (H2O, s).Embodiment 6: succinimido paraacetaminobenzoic acid (NHS-ester II sees above-mentioned sequence III) synthetic

5.375g acetaminobenzoic acid (0.03 mole), 8.453g DSC (0.033 mole) and 150THF are placed flask.Mixed solution is in stirring at room more than 30 minutes.Add the 5mL triethylamine, this reactant stirred under room temperature 16 hours.Remove with the rotary evaporation method and to desolvate.The solid of gained washes three times (under the vacuum filtration conditions) with water to remove by product.With solid vacuum-drying, obtain about 6.0 pairs of acetaminobenzoic acids (NHS-ester II), productive rate 72%.NMR result is as follows: ¹H NMR (400MHz, DMSO-d ₆) δ ppm2.12 (3H, s), 2.89 (4H, s), 3.36 (H2O, s), 7.84 (2H, m), 8.05 (2H, m).Embodiment 7: acetyl pronestyl bridged piperazine derivatives (APP) and APP acid synthetic

1.69g the BAP of purifying and 10mL THF place the flask of a 25mL.0.687g succinimide is dissolved among the THF of 29mL acetaminobenzoic acid.Second kind of solution slowly stirs and adds in the BAP solution.Reaction stirred, and with thin-layer chromatography monitoring (TLC) (100% ethyl acetate is as solvent of TLC) until monitoring less than succinimide to acetaminobenzoic acid.Form a large amount of solids.Remove with transfer pipet and to desolvate.Solid washs three times (10mL * 3) to remove unnecessary BAP with ether.Solid vacuum-drying obtain 66% product (APP, 550mg).

166.5mg APP and 5mL diethylformamide (DMG) place the flask of a 50mL, add 69.56 μ L triethylamines then.The succinyl oxide of 50mg is dissolved among the 2mL DMF, and under agitation slowly adds APP solution.Reaction is at room temperature stirred and was carried out 1 hour.Add 20mL ether (Et ₂O) after, reaction mixture places refrigerator overnight with precipitated product (APP acid).Remove with transfer pipet and to desolvate, and with ether washed twice (5mL * 2).Remaining solid is obtained the APP acid of about 100mg, productive rate 46% by vacuum-drying.Synthesizing of embodiment 8:NAPA protein conjugates

Synthesizing of a.APP-OBt ester

1. weighing 60.00mg (0.138 mole) APP acid and 16.54mg (0.152 mole) HOBt, and place the bottle of a 2mL.Put into magnetic stirring bar.The dry DMF of 600 μ l adds bottle with dissolving APP acid and hydration hydroxybenzotriazole (HOBt) with transfer pipet.

2. in the above-mentioned solution of weighing 28.54mg (0.138 mmole) dicyclohexyl carbonyl diurethane imines (DDC), and stirring adding.

3. reaction is at room temperature stirred and was carried out 2-3 hour.So the acid after the activation can be used to and albumen coupling.

Synthesizing of b.APP-protein conjugates

Protein solution

A.0.15M NaHCO ₃In ovalbumin

1. weighing 0.315g sodium bicarbonate and adding in the 25.0mL volumetric flask is dissolved to lucky 25.0mL with deionized water then.

2.50mg ovalbumin be dissolved in 8mL 0.15M NaHCO at the bottle that screw-cap is arranged and put into magnetic stirring bar of a 16mL ₃(sodium bicarbonate).

The B.KLH aqueous solution

50mg KLH is dissolved in the 8mL deionized water in the centrifuge tube of a 16mL, and stirs gently in cold house (4 ℃) and spend the night.Then that this centrifuge tube is centrifugal.Supernatant liquor is poured in the screw-topped bottle of a 16mL into (reaction vessel that will be used as synthetic binding substances) and deposited in the refrigerator.

The C.APP-binding substances

1. above-mentioned each the APP-OBt DMF solution of 300 μ l that makes from above-mentioned steps adds KLH water (2.B) and the ovalbumin damping fluid (2.A) respectively with transfer pipet and stirs.Reaction was at room temperature carefully stirred 10 minutes, spent the night in the cold house that puts into 4 ℃ then.

2. above-mentioned two kinds of conjugated protein solution of APP are to deionized water dialysis three times, then to phosphate buffered saline buffer (PBS) dialysis three times (the ovalbumin binding substances uses 6-8, the dialysis tubing of 000MW scope).

The synthetic embodiment 9:BOC-BA of pronestyl bridged piperazine derivatives and protein conjugates thereof and BOC-SB's is synthetic

A. prepare BOC-BA

1. weighing 3.425g benzaminic acid (ABA) and 1.5g sodium hydroxide, and place the flask of a 100mL.

2. stir and add 25mL water.

3. add 5.995g two tert.-butoxy heavy carbonic esters (DBDC).

4. reaction is at room temperature stirred and is spent the night.

Solution with the 1N hcl acidifying to pH4-5 (needing 37.5mL 1N hydrochloric acid approximately).

6. product ethyl acetate extraction three times (3 * 25mL).

7. the washing of ethyl acetate (EtOAc) solution with water is three times (3 * 25mL).

8.EtOAc solution dried over mgso.

9. remove with the rotary evaporation method and desolvate.

10. product is weighed (W), and productive rate calculates according to following formula:

Y＝(W/5.01)×100％

B.BOC-SB is synthetic

1. weighing 3.555g BOC-BA (0.015 mole) and be dissolved in the 50mL acetonitrile.

2. add 4.224g DSC (0.015 * 1.1 mole) and 5.22mL TEA (0.015 * 2.5 mole) to this mixture.

3. reaction is at room temperature stirred and is carried out 2-3 hour (reactant 50%EtOAc and 50% hexane inspection).

4. remove and desolvate.

5. degree of purity of production detects with TLC (with chromatographic column this product of purifying repeatedly).

6. product is weighed (W), and productive rate calculates according to following formula

Y＝(W/5.925)×100％

NMR result is as follows: ¹H NMR (400MHz, DMSO-d ₆) δ ppm1.50 (9H, s), 2.88 (4H, s), 3.34 (H2O, s), 7.71 (2H, m), 8.00 (2H, m).Synthesizing of embodiment 10:BOC-PP and BOC-PP-NHS ester

A.BOC-PP's (tert.-butoxy formamyl pronestyl bridged piperazine derivatives) is synthetic

1. 0.688mg BAP is dissolved in 10mL THF.

2. 334mg BOC-SB is dissolved in 15mL THF.

3. the solution of step 2 (BOC-SB) slowly stirs in the solution (BAP) that adds step 1.Produce some solid precipitations after 5-10 minute.

4. reaction was at room temperature stirred more than 30 minutes.

5. remove with transfer pipet and desolvate.Solid washs three times (10mL * 3) to remove unnecessary BAP with ether.

6. with solid vacuum-drying, obtain 87% product (BOC-PP, 340mg).

BOC-PP is moisture absorption very easily, should deposit in the moisture eliminator.

Synthesizing of b.BOC-PP-NHS ester

1. weighing 107.92mg (0.276 mmole) BOC-PP and place a 4mL bottle.Put into a magnetic stirring bar.With transfer pipet 500 μ l dry DMF and 40 μ l TEA are added in the bottle with dissolving BOC-PP.

2.27.6mg (0.276 mmole) succinyl oxide is dissolved among the 100 μ l DMF, and stirs in the solution that adds step 1; Bottle is with 100 μ l DMF solvent cleaning, and joins in the solution to guarantee quantitative transfer.

3. reaction is at room temperature stirred and was carried out 1 hour.

4. weighing 77.78mg DSC (0.152 mmole) and adding in the above-mentioned reaction solution stirs afterwards and adds 56.0 μ l TEA.

5. reaction is at room temperature stirred and was carried out 2 hours.Embodiment 11: pronestyl protein conjugates synthetic

A. protein solution

A.0.15M NaHCO ₃In ovalbumin

1. weighing 0.630g sodium bicarbonate and adding in the volumetric flask of a 50.0mL is dissolved to lucky 50.0mL with deionized water then.

2.100mg ovalbumin a 40mL screw-cap is arranged and add magnetic stirring bar the bottle in be dissolved in 16mL 0.15M NaHCO ₃(sodium bicarbonate).

The B.KLH aqueous solution

100mg KLH is dissolved in the 16mL deionized water in a centrifuge tube, stir gently in 4 ℃ cold house and spend the night.This centrifuge tube is centrifugal, then supernatant liquor is poured in the screw-topped bottle of a 40mL into (reaction vessel that will be used as synthetic binding substances) and deposited in the refrigerator.

B.BOC-PP-NHS combines with proteic

The above-mentioned BOC-PP-NHS DMF of the 400 μ l solution (BOC-PP-NHS of 0.138 mmole) that makes from embodiment 6b.5 goes to KLH water and the ovalbumin buffer soln with transfer pipet, and stirs.At room temperature careful stirring of reaction carried out 10 minutes, deposited in then in cold house's (4 ℃) and spent the night.

The going of c.BOC-group protected the purifying with binding substances

1. two kinds of protein solutions that combine BOC-PP-NHS are to deionized water dialysis three times.

2. two kinds of protein solution freeze-drying in a vacuum (frost drying) are to obtain egg white solid.Every kind of albumen adds 5mL CH ₂Cl ₂, add the 5mL trifluoroacetic acid afterwards.Stir after 5 minutes, solvent concentrates in a vacuum, and residuum is dissolved in 16mL PBS damping fluid again.The muddy liquid of gained is to deionized water dialysis three times, again to PBS damping fluid dialysis three times (using 6-8, the dialysis tubing of 000MW scope).

Reactive derivatives of the present invention provides the medicine that will have dialkyl amino group or other compound to be attached to new compound on albumen or other carrier compound.Many medicines with dialkyl amino group can benefit from derivative of the present invention.The method that two kinds of different types of drugs is derivatized to reactive derivatives of the present invention has above been described.One of skill in the art will be appreciated that other method that can use other dialkyl amino based compound of deriving, and these methods are can be with different compounds different, but deriving technology belongs to the method for this area accurately.What be used for that method that functional group with reactive piperazine derivative N-terminal or difunctional spacer is attached to carrier compound can be with medicine and carrier compound combination is different and different, but also complete in the technical scope of this area.

Claims (22)

1. one kind is used for and carrier compound bonded component, and it comprises:

Wherein D is the compound that does not have dialkyl amino group at it in the derived structure, and this dialkyl amino group is by this component

Residue substitutes, and n is the integer more than or equal to 1.

2. the component in the claim 1, wherein D is the medicine that is selected from lignocaine, pronestyl, N-acetyl pronestyl, disopyramide, chloroquine, diphenhydramine, methadone, imipramine, Desipramine, amitriptyline, noxiptiline and nortriptyline.

3. the component in the claim 1, wherein terminal nitrogen is attached on the carrier compound.

4. the component in the claim 3, wherein carrier compound is a proteinaceous substances.

5. the component in the claim 3, wherein carrier compound is attached on the solid support.

6. the component in the claim 5, wherein solid support is a polymer beads.

7. the component in the claim 1, it also comprises the difunctional spacer that is attached on the terminal nitrogen.

8. the component in the claim 7, wherein difunctional spacer have with terminal nitrogen bonded first function of piperazine derivative terminal and with the carrier compound bonded second function end.

9. the component in the claim 8, wherein difunctional spacer is selected from cyclic anhydride, two N succinimide radical derivative and dialdehyde.

10. the component in the claim 8, wherein carrier compound is a proteinaceous substances.

11. the component in the claim 8, wherein carrier compound is attached on the solid support.

12. the component in the claim 11, wherein solid support is a polymer beads.

13. an immunogen, it comprises:

A kind of being selected from With

Component, wherein D is the medicine that does not have dialkyl amino group at it in the derived structure, this dialkyl amino group is by this compound

Derivative substitutes, and n is the integer more than or equal to 1, and X is difunctional spacer.

14. the immunogen in the claim 13, its Chinese traditional medicine are selected from lignocaine, pronestyl, N-acetyl pronestyl, disopyramide, chloroquine, diphenhydramine, methadone, imipramine, Desipramine, amitriptyline, noxiptiline and nortriptyline.

15. the immunogen in the claim 13, wherein difunctional spacer are selected from cyclic anhydride, two N succinimide radical derivative or dialdehyde.

16. the immunogen in the claim 13, wherein n is from 1 to 3 integer.

17. the immunogen in the claim 13, wherein albumen is selected from ovalbumin, lockhole limpet hemocyanin and bovine serum albumin.

18. a particle reagents, it comprises:

(a) have the polymer beads of kernel and shell, wherein kernel is when the sodium D-line wavelength measurement, have the polymkeric substance that is not less than 1.54 specific refractory power, and shell is following polymer of monomers:

(i) two key unsaturated monomers, it has the functional group of nucleophilic compound reaction that can be relevant with biology;

(ii) Ren Xuan other pair key unsaturated monomer, its content is enough to produce the insoluble polymer particle, and

(iii) be no more than the interior nuclear monomer of shell 10 weight parts, shell is to form by polyreaction in the presence of covalently bound kernel;

(b) a kind of component, it is selected from:

With Wherein D is the medicine that does not have dialkyl amino group at it in the derived structure, and this dialkyl amino group is by component

Derivative replaces, and n is the integer more than or equal to 1, and X is difunctional spacer.

19. the particle reagents in the claim 18, its Chinese traditional medicine are selected from lignocaine, pronestyl, N-acetyl pronestyl, disopyramide, chloroquine, diphenhydramine, methadone, imipramine, Desipramine, amitriptyline, noxiptiline and nortriptyline.

20. the particle reagents in the claim 18, wherein difunctional spacer are selected from cyclic anhydride, two N succinimide radical derivative and dialdehyde.

21. the particle reagents in the claim 18, wherein n is from 1 to 3 integer.

22. the particle reagents in the claim 18, wherein carrier compound is selected from bovine serum albumin, polyethylenepolyamine and human serum albumin.