CN110294789A - The synthetic method of the oligopeptides containing DOPA and its application in terms of anti-Parkinson's disease prodrug - Google Patents
The synthetic method of the oligopeptides containing DOPA and its application in terms of anti-Parkinson's disease prodrug Download PDFInfo
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Abstract
The invention discloses the synthetic methods of the oligopeptides containing DOPA and its application in terms of anti-Parkinson's disease prodrug, synthetic route to be
Description
Technical field
The present invention relates to a kind of synthetic method of peptide and applications, the in particular to synthetic method and application of the oligopeptides containing DOPA.
Background technique
Parkinson's disease is to seriously threaten the global disease of middle-aged and the old's life and health.L-DOPA is always to treat pa
The gloomy disease of gold is most effective, and most widely used drug is the gold standard in similar drugs.Levodopa have in physiological conditions compared with
Large scale be in the form of uncharged neutral molecule existing for, thus blood-brain barrier (Blood Brain can be passed through
Barrier, BBB) enter brain, it then is degraded into dopamine under the action of fragrant decarboxylase, to restore brain due to disease
Reduced dopamine concentration.After oral, levodopa is by the stomach of highly acid and the small intestine rich in digestion degrading enzyme, about
85% is converted to dopamine, and only 15% amount can pass through amino acid transport channel (Amino Acid Transport
System amino acid carrier (AT)) enters blood circulation.DOPA is after amino acid carrier is absorbed into human body, outside
It is largely decomposed again under all decarboxylations, finally the oral dose of only 1-3% can work into brain.This is caused
Abnormal low of the bioavailability of DOPA (bioavailability).
By by levodopa and thermophilic chromium tumor noble cells (PC12) cell of rat adrenal medulla, pheochromocytoma cells
Or midbrain dopaminergic neuron co-cultures confirmation levodopa in vitro to DA serotonergic neuron and other neurons with latent
Neurotoxic effect, and this toxic effect and drug dose, concentration and action time are closely related.There are also poles for DOPA
Bad toxic side effect, after using 3~5 years, many patients can suffer from various complication, such as dyskinesia, myasthenia etc..This
When levodopa do not worked to these patients, be referred to as Parkinson's agent end phenomenon.
Human small intestine's wall not only has the channel for absorbing unimolecule amino acid, and there are also directly absorb oligopeptides (dipeptides or tripeptides)
Oligopeptides channel (Oligopeptide Transporter), thus the oligopeptides containing DOPA can not need to be broken down into single amino
Acid and be directly entered human body.Early in 1974, Felix etc. synthesized more than ten straight chain dipeptides and tripeptides containing DOPA, found it
Middle dipeptides (DOPA-Lys, Val-DOPA, Tyr-DOPA, Glu-DOPA) and tripeptides (Gly-Gly-DOPA, Gly-DOPA-Gly,
Pro-DOPA-DOPA) all than the L-DOPA of equivalent molar number in terms of alleviating catatonia caused by reserpine a kind of (anti-pa
The gloomy sick efficacy testing model of gold) it is more effective.2000, Cingolani etc. modified the amino of L-DOPA using diketone morpholine ring
With carboxyl, research finds that the derivative can be absorbed by gastrointestinal tract and release L-DOPA after being digested in blood of human body.
2013, Zhou etc. reported the synthesis and anti-Parkinson's disease active testing of more than ten dipeptides of straight chain containing DOPA, found more than one
Bar there is outstanding anti-Parkinson's disease activity with the straight chain dipeptidase derivant of unnatural amino acid.Contain a small amount of D- type in human body
Amino acid, the oligopeptides being made of D- type and L-type amino acid is due to lacking the enzyme of specificity and the speed that is degraded is very slow
Slowly.Nineteen ninety-five, Wang etc. report the synthesis of tripeptides D-p-hydroxyphenylglycine-L-Pro-L-DOPA, experiment hair
Now the L-DOPA prodrug can reduce the circling behavior (one of the mouse of the nigrostriatal lesion of crystal methamphetamine induction significantly
Kind anti-Parkinson's disease drug effect test model).2013, Wang etc. synthesized the more of one kind phenylglycine containing dextrorotation (PhGly)
Bar dipeptides, H-D-PhGly-L-DOPA-OH.Various experiments confirm that D- the and L- mixed type dipeptides is easier to than levodopa by small
Intestinal wall enters blood circulation, has stronger anti-Parkinson's disease activity, while also having slow release effect.
Cyclic dipeptides containing DOPA have unique bioactivity due to L-DOPA.Early in 2000,
The research discovery mangrove ascidian ecteinascidia turbinata of Jeedigunta etc. can use cyclo (DOPA-
DOPA important anticancer chemotherapeutic agent Ecteinascidin 743 (ET 743)) is synthesized in vivo as intermediate, for the drug
Biosynthesis provides an important approach.2008, the trials such as Zhang Chongjing synthesized cyclo (DOPA-Pro) as novel
The peptidomimetic inhibitor of peptidyl-prolyl isomerase Pin1, but finally the Cyclic dipeptides are not provided to the specific rejection ability number of Pin1
According to.2017, the researchs such as Manchineella discovery cyclo (DOPA-DOPA) and cyclo (DOPA-Phe) were shown well
Remove free radical and oxidation resistant ability.More importantly author has been experimentally confirmed the two Cyclic dipeptides containing DOPA pair
PC12 cell is all without cytotoxicity.
The latest data retrieved according to Scifinder: it has been reported that the Cyclic dipeptides one containing DOPA share 4 kinds, they
It is cyclo (L-DOPA-L-DOPA), cyclo (L-DOPA-L-Tyr), cyclo (L-DOPA-L-Phe), cyclo (L- respectively
DOPA-D/L-Pro) (mixture of two kinds of isomers), cyclo (L-DOPA-Gly).It has been reported that contain DOPA skeleton, but
Catechol therein is by about more than 20 kinds of derivative Cyclic dipeptides, such as cyclo (L-DOPA (Me2)-Gly), cyclo (L-
DOPA(CH2)-Gly).Since in these synthesis processes, the catechol group of DOPA is unprotected, the former directly reacts
To dopa methyl ester easily aoxidize, purification is complicated, and the latter has used other amino acid methyl ester hydrochlorides and DOPA to be reacted
Mode is a kind of improvement.The reaction of the two is required in 100 DEG C or so lower back flow reactions, still exist partial amino-acid due to
Esterification reaction of organic acid is more complicated, is heated to reflux the reasons such as unfavorable to thermo-responsive protecting group to part, therefore its applicability is relatively narrow.It is micro-
Wave radiation is harsh compared with the condition of high-temperature heating, and the racemization of amino acid is easily caused in reaction process, obtained mixing
Object is difficult to isolate and purify.Second method: raw material is done using the DOPA straight chain dipeptides containing the catechol group protected to prepare
The derivative of Cyclic dipeptides containing DOPA.Aicher etc. has synthesized cyclo (L-DOPA (Me with two methyl protection phenolic hydroxyl groups2)-Gly),
However the limitation of methyl protection is larger, the ehter bond of generation is difficult to be broken off, and the reaction condition of deprotection is harsher, laboratory
Operate it is relatively difficult, therefore not yet unprotected Cyclic dipeptides containing DOPA synthesize in this way.In conclusion now
The Cyclic dipeptides containing DOPA synthesized only have 4 kinds, are especially the failure to synthesize the ring containing DOPA containing active side chain amino acid two
Peptide.
The polypeptide containing L-DOPA is prepared using based on Solid-phase synthesis peptides (SPPS) method of Fmoc, it is necessary first to which synthesis is closed
Key intermediate Fmoc-DOPA (Acetonide)-OH.The compound is expensive, and every gram of price is up to 10,000 yuan, and is not easy to purchase
It buys.2008, Liu et al. reported a kind of simple method for synthesizing of intermediate: first using N- carbon ethoxybenzene phthalimide
(N-Carboethoxy Phthalimide, CE-Phth) reagent the amido protecting of L-DOPA at phthalimide,
It is secondary to use MeOH/SOCl2The converting carboxylate groups of L-DOPA at methyl esters, this measure avoids Pictet-Spenglar reactions pair
The interference of contracting acetone cyclization in next step；Then, freshly prepd Phth-DOPA-OMe and 2,2-dimethoxypropane
(dimethoxypropane, DMP) and catalyst p-methyl benzenesulfonic acid (TsOH) flow back in anhydrous benzene together, are prepared for Phth-
DOPA(Acetonide)-OMe；Then, using the Phth group of hydrazine deprotection amido, using LiOH hydrolysising protection carboxyl
Methyl esters generates intermediate product H-DOPA (Acetonide)-OH；The latter reacts with Fmoc-OSu, generates target product Fmoc-
DOPA(Acetonide)-OH.2012, Messersmith etc. advanced optimized Fmoc-DOPA in United States Patent (USP)
(Acetonide) synthetic method of-OH: discovery replaces Phth to protect the amino of L-DOPA same using trifluoroacetyl group (Tfa)
Sample can be to avoid Pictet-Spenglar side reaction, since Tfa protecting group can be cut with methyl esters in LiOH solution
It removes, therefore the synthesis of Fmoc-DOPA (Acetonide)-OH is reduced into 5 steps from 6 steps, yield, which also improves, to be connect by about one time.The conjunction
It can also be used to prepare Boc-DOPA (Acetonide)-OH, H-DOPA (acetonide)-OMe, H-dopamine at strategy
(Acetonide) etc. the catecholamine analog derivative of contracting acetone protection.Wherein H-dopamine (Acetonide) be used for
Docosahexaenoic acid (being commonly called as docosapentaenoic acid, docosahexaenoic acid, DHA) is reacted, and high-purity is successfully prepared
Anti-Parkinson dopamine prodrug DHA-dopamine.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of pervasive sides for synthesizing the Cyclic dipeptides containing DOPA
Method.
Another object of the present invention is a kind of using intermediate obtained by the above method in providing.
It is yet a further object of the present invention to provide the applications of the dipeptides containing DOPA.
The technical solution used in the present invention is:
A kind of liquid-phase synthesis process of the dipeptides containing DOPA, synthetic route are as follows:
In formula, R1And R2In one be protection DOPA side chain, preferred protecting group be contracting acetone, another for protect
Or unprotected amino acid side chain；
R5For amino protecting group, R6For carboxyl-protecting group；Synthetic method includes the following steps:
It willIt is dissolved in organic solvent A, condensing agent is added, liquid phase condensations react
To straight chain dipeptides R5-AA1-AA2-OR6；
By straight chain dipeptides R5-AA1-AA2-OR6Deprotection obtains straight chain dipeptides；Or by straight chain dipeptides R5-AA1-AA2-OR6It is molten
Solution is reacted in alkalinity deprotection liquid, and liquid phase reactor sloughs amino protecting group, the Cyclic dipeptides containing DOPA protected
In some examples of the invention, acid deprotection liquid removing DOPA Side chain protective group is further used, is contained
The Cyclic dipeptides containing DOPA of free catechol group.Acidity deprotection liquid is selected from the solution containing TFA, HCl.
In some examples of the invention, R1、R2Be independently selected from L-type or D type Gly, Ala, Val, Leu, Ile, Phe, Trp,
The amino acid side chain of Tyr, Asp, Asn, Glu, Lys, Gln, Met, Ser, Thr, Cys, Pro, His, Arg or DOPA.
In some examples of the invention, R5Selected from Fmoc, Boc；R6Selected from methyl, tert-butyl.
In some examples of the invention, the solvent that organic solvent A, alkalinity are deprotected liquid is independently selected from acetonitrile, N, N- bis-
Methylformamide (DMF), methylene chloride (DCM), ethyl acetate (EA) are at least one.
In some examples of the invention, the alkaline components in alkalinity deprotection liquid are selected from piperidines, piperazine.Further
, mass percentage of the piperidines in alkaline solution is 5~60%, preferably 15~50%.
The new intermediate product or final product that above-mentioned liquid-phase synthesis process synthesizes.
Dipeptides containing DOPA prepare L-DOPA prodrug, anti-Parkinson's disease sustained release prodrug in application, in which:
The structural formula of Cyclic dipeptides containing DOPA are as follows:
The structural formula of straight dipeptides containing DOPA are as follows:
In formula, R1For L-type or the side chain of D type amino acid；
Preferably, the middle R of the straight dipeptides containing DOPA1For L-type or the side of D type Gly, Leu, Val, Phe, Ile, Tyr or Asp
Chain；
Preferably, the middle R of the Cyclic dipeptides containing DOPA1For L-type or D type Gly2, Ala, Trp, Lys, Glu, Gln, Ser,
The side chain of DOPA.
In some examples of the invention, R1Selected from L-type or D type Gly, Ala, Val, Leu, Ile, Phe, Trp, Tyr,
The amino acid side chain of Asp, Asn, Glu, Lys, Gln, Met, Ser, Thr, Cys, Pro, His, Arg or DOPA.
The beneficial effects of the present invention are:
In the synthesis process, the intermediate of Cyclic dipeptides containing the DOPA cyclo of contracting acetone protection is prepared in the method for the present invention
(DOPA (acetonide)-AA) has good stability, can save steadily in the long term, simultaneously, it is only necessary to which a step can turn
The Cyclic dipeptides containing DOPA are turned to, the structural instability of Cyclic dipeptides containing DOPA is overcome, is unable to the deficiency of long-term preservation.
The method of the present invention has good universality, overcomes the deficiency for needing back flow reaction in the prior art, reacts item
Part is mild, side reaction is less, and the cost of raw material is more cheap, and the purity of product is higher and is easily isolated.
The Cyclic dipeptides containing DOPA that the method for the present invention is prepared have good stability, anti-degradation energy in liver homogenate
Power is strong, compared with L-DOPA, has better stability, is expected to be developed into that bioavilability is high, long-acting Parkinson's disease is controlled
It treats straight chain dipeptides ratio DOPA-Met carboxylic-containing acid such as drug, especially DOPA-Asp etc. to stablize, more suitable for doing anti-Parkinson
The L-DOPA prodrug of disease uses.Simultaneously as its bioavilability greatly improves, it is expected to that L-DOPA treatment Parkinson is greatly reduced
The side effect of disease.
Detailed description of the invention
Fig. 1 is the liver homogenate degradation half life result of the dipeptides of straight chain containing DOPA；
The liver homogenate degradation half life result of Fig. 2 Cyclic dipeptides containing DOPA.
Specific embodiment
A kind of liquid-phase synthesis process of the dipeptides containing DOPA, synthetic route are as follows:
In formula, R1And R2In one be protection DOPA side chain, preferred protecting group be contracting acetone, another for protect
Or unprotected amino acid side chain；
R5For amino protecting group, R6For carboxyl-protecting group；Synthetic method includes the following steps:
It willIt is dissolved in organic solvent A, condensing agent, liquid phase condensations reaction is added
Obtain straight chain dipeptides R5-AA1-AA2-OR6；
By straight chain dipeptides R5-AA1-AA2-OR6Deprotection obtains straight chain dipeptides；Or by straight chain dipeptides R5-AA1-AA2-OR6It is molten
Solution is reacted in alkalinity deprotection liquid, and liquid phase reactor sloughs amino protecting group, the Cyclic dipeptides containing DOPA protected
Amino acid side chain can be protected according to the power of its reactivity using general method, or without protecting
Shield.The type of amino acid can be selected accordingly without particular/special requirement according to its special-purpose.From the extensive of raw material sources
Property, ready availability and product safety etc. consider, the preferred L-type of amino acid side chain or D type Gly, Ala, Val, Leu, Ile,
The amino acid side of Phe, Trp, Tyr, Asp, Asn, Glu, Lys, Gln, Met, Ser, Thr, Cys, Pro, His, Arg or DOPA
Chain.
Liquid reactive temperature carries out at room temperature without particular/special requirement, and temperature increases the progress for being conducive to reaction.For
Reaction is kept uniformly to carry out in the liquid phase, reaction temperature is maintained under the boiling point of solvent.Such as amino acid side chain protecting group
It is sensitive to reaction temperature, it can be reacted below sensitive temperature, to reduce the generation of impurity, reduce the difficulty of later separation
Degree.
Reacting the solvent used includes but is not limited to the common solvents such as DCM, CAN, EA, DMF, NMP.It is preferable molten to obtain
Xie Du keeps reaction more uniform.
The condensing agent that condensation reaction uses can be depsipeptides agent commonly used in the art, including but not limited to EDC, DCC,
The common condensing agent for forming peptide bond such as HBTU, HATU.
As the further improvement of above-mentioned liquid-phase synthesis process, further uses acid deprotection liquid removing DOPA side chain and protect
It protects base (contracting acetone), obtains the Cyclic dipeptides containing DOPA containing free catechol group.Acidity deprotection liquid is selected from containing the molten of TFA, HCl
Liquid.
Amino protecting group and carboxyl-protecting group can be protecting group commonly used in the art.As above-mentioned liquid-phase synthesis process
It is further improved, R5Selected from Fmoc, Boc；R6Selected from methyl, tert-butyl.Schematically synthetic route is respectively
As the further improvement of above-mentioned liquid-phase synthesis process, the solvent that organic solvent A, alkalinity are deprotected liquid is independently selected from
Acetonitrile, N,N-dimethylformamide (DMF), methylene chloride (DCM), ethyl acetate (EA) are at least one.
As the further improvement of above-mentioned liquid-phase synthesis process, the alkaline components in alkalinity deprotection liquid are selected from piperidines, piperazine
Piperazine.Further, mass percentage of the piperidines in alkaline solution is 5~60%, preferably 15~50%.
Below with reference to embodiment, technical solution of the present invention is further illustrated.
Two kinds of synthetic method introductions of Cyclic dipeptides containing DOPA:
The synthesis of the Cyclic dipeptides containing DOPA is divided into the progress of three steps:
1) straight chain dipeptides is synthesized: using the amino acid and amino acid methyl ester of Fomc protection in dry DCM solvent, in carbon
It is the protected straight chain dipeptides Fmoc-AA of side chain that diimine type condensing agent DCC, which acts on lower dehydrating condensation,1-AA2-OMe；
2) synthesis of Cyclic dipeptides is protected: and then this straight chain dipeptides is sloughed into Fmoc group under alkaline condition while occurring to divide
Sub- internal condensation reacts to form Cyclic dipeptides；
3) finally at high concentration TFA, side chain protecting group and DOPA catechol group the deprotection of Cyclic dipeptides: are sloughed
On contracting acetone protecting group, obtain target Cyclic dipeptides containing DOPA.
Using the type of DOPA intermediate as differentiation foundation, then this synthetic route can be divided into two branch lines
That is: synthesis Cyclic dipeptides are 1. reacted with the common amino acid methyl esters of purchase with DOPA intermediate Fmoc-DOPA (Acetonide)-OH；
2. synthesizing Cyclic dipeptides with the fmoc-protected common amino acid of purchase using DOPA intermediate H-DOPA (Acetonide)-OMe.
Specific synthetic route selects amino acid methyl ester price and fmoc-protected amino acid price according to accordingly buying to integrate
Consider.
The synthesis of straight chain methyl dipeptide: synthetic route is as follows:
By fmoc-protected conventional amino acid or Fmoc-DOPA (Acetonide)-OH and amino acid methyl ester or H-
DOPA (Acetonide)-OMe makees solvent in DMF, and the mode that HBTU+DIEA makees condensing agent is condensed into straight chain dipeptides Fmoc-AA1-
AA2-OMe。
Protect the synthesis of Cyclic dipeptides: synthetic route is as follows:
By straight chain dipeptides Fmoc-AA1-AA2- OMe, which is placed in the DMF solution of 20% piperidines, to react, and Fmoc protecting group will be taken off
It removes and forms Cyclic dipeptides.
The Cyclic dipeptides synthesized by Fmoc-DOPA (Acetonide)-OH
Synthetic route by the Cyclic dipeptides of Fmoc-DOPA (Acetonide)-OH synthesis is as follows
In formula, a) DIEA, HBTU b) piperidines, ACN c) TFA, TIS, H2O；R1=Gly, Ala, Leu, Glu, Lys, Ser,
Trp residue.
Pass through the synthesis of the Cyclic dipeptides of H-DOPA (Acetonide)-OMe synthesis
The synthesis of embodiment 1, Cyclo (DOPA-Ala) (with the Cyclic dipeptides formed containing simple side chain amino acid)
Take Fmoc-DOPA (Acetonide)-OH (2.5mmol, 1.15g) and H-Ala-OMeHCl (2.5mmol,
It 0.35g) is dissolved in DMF (20ml), is added DIEA (5mmol, 0.83ml), solution is in room temperature with HBTU (2.5mmol, 0.95g)
Then lower stirring 24 hours is evaporated in vacuo and removes solvent to a small amount of.Saturation NaHCO is added in gained remnants3Aqueous solution and acetic acid
In ethyl ester (1:1), with saturation NaHCO3Aqueous solution washs 5 times.Then, by organic phase anhydrous Na2SO4Dry, filter removing
Na2SO4, solvent is removed in vacuum, is recrystallized with a small amount of ethyl acetate and n-hexane, obtains straight chain dipeptides Fmoc-DOPA
(Acetonide)-Ala-OMe (1.07g, 79%).Fmoc-DOPA(Acetonide)-Ala-OMe(M:C31H32N2O7)([M+
H]+:Calc.544.2210Found 544.2230).
Straight chain dipeptides Fmoc-DOPA (Acetonide)-Ala-OMe (1mmol, 0.54g) is suspended in 20% piperidines/ACN
In (25ml) solution and stir 24 hours.Gained suspension is concentrated in vacuo and is washed 5 times with ether.The white solid that will be obtained
It is put into drying in vacuum oven, obtains corresponding pure products Cyclic dipeptides.Cyclo [DOPA (Acetonide)-Ala] (0.24g,
83%).Cyclo[DOPA(Acetonide)-Ala](M:C15H18N2O4)([M+H]+:Calc.291.1339.Found
291.1569).
Cyclic dipeptides Cyclo [DOPA (Acetonide)-Ala] (1mmol, 0.28g) with protection is added to the TFA of 25ml:
TIS:H2In the mixed liquor of O=(95:2.5:2.5), 1h is stirred, is then precipitated to obtain solid with ice ether, be centrifuged (4 DEG C,
Obtained white solid is put into drying in vacuum oven after 8000rpm), obtains corresponding pure products Cyclic dipeptides Cyclo
(DOPA-Ala) (0.24g, 94%).Cyclo(DOPA-Ala)(M:C12H14N2O4)([M+H]+:Calc.251.1026Found
251.1013)。
The synthesis of embodiment 2, Cyclo (DOPA-Ser) (with the Cyclic dipeptides formed containing polar side chains)
(1) synthesis of compound Fmoc-DOPA (Acetonide)-Ser (tBu)-OMe
Take Fmoc-DOPA (Acetonide)-OH (2.5mmol, 1.15g) and H-Ser (tBu)-OMeHCl
(2.5mmol, 0.45g) and HBTU (2.5mmol, 0.95g) is dissolved in DMF (100ml), is added DIEA (5mmol, 0.83ml),
Solution is stirred at room temperature 24 hours, is then evaporated in vacuo and removes solvent to a small amount of.Saturation NaHCO is added in gained remnants3Water
In solution and ethyl acetate (1:1), with saturation NaHCO3Aqueous solution washs 5 times.Then, by organic phase anhydrous Na2SO4It is dry,
It is filtered to remove Na2SO4, solvent is removed in vacuum, is washed with a small amount of ethyl acetate, filters, obtains straight chain dipeptides Fmoc-DOPA
(Acetonide)-Ser (tBu)-OMe (1.03g, 67%).Fmoc-DOPA(Acetonide)-Ser(tBu)-OMe(M:
C35H40N2O8)([M+H]+:Calc.617.2857Found 617.2899).
(2) synthesis of compound Cyclo [DOPA (Acetonide)-Ser (tBu)]
Straight chain dipeptides Fmoc-DOPA (Acetonide)-Ser (tBu)-OMe (1mmol, 0.62g) is suspended in 20% piperazine
In pyridine/ACN solution (50ml) and stir 24 hours.Gained suspension is concentrated in vacuo and is precipitated and is washed 5 times with ether.Will
To white solid be put into vacuum oven dry, obtain corresponding pure products Cyclic dipeptides Cyclo [DOPA (Acetonide)-
Ser (tBu)] (0.27g, 75%).Cyclo[DOPA(Acetonide)-Ser(tBu)](M:C19H26N2O5)([M+H]+:
Calc.363.1914.Found 363.1833).
(3) synthesis of compound Cyclo (DOPA-Ser)
25ml is added in Cyclic dipeptides Cyclo [DOPA (Acetonide)-Ser (tBu)] (1mmol, 0.36g) with protection
TFA:TIS:H2In the mixed liquor of O=(95:2.5:2.5), 1h is stirred, is then precipitated to obtain solid, centrifugation (4 with ice ether
DEG C, 8000rpm) after obtained white solid is put into vacuum oven dry, obtain corresponding pure products Cyclic dipeptides Cyclo
(DOPA-Ser) (0.26g, 96%).
Cyclo(DOPA-Ser)(M:C12H14N2O5)([M-H]-:Calc.265.0830Found 265.0876).1H NMR
(400MHz,TFA/DMSO)δ6.96–6.86(m,2H),6.73(dd,J1=8.1, J2=2.2Hz, 1H), 4.66 (dt, J1=
10.7,J2=5.0Hz, 1H), 4.55-4.31 (m, 1H), 3.83 (dd, J1=12.2, J2=3.8Hz, 1H), 3.57-3.42 (m,
1H),3.28–3.10(m,2H).
The synthesis (Cyclic dipeptides formed with Dextrorotatory amino acids) of embodiment 3, Cyclo (DOPA-D-Glu)
(1) synthesis of compound Fmoc-DOPA (Acetonide)-D-Glu (OtBu)-OMe
Take H-DOPA (Acetonide)-OMeHCl (2.5mmol, 0.72g) and Fmoc-D-Glu (OtBu)-OH
(2.5mmol, 1.06g) and HBTU (2.5mmol, 0.95g) is dissolved in DMF (100ml), is added DIEA (5mmol, 0.83ml),
Solution is stirred at room temperature 24 hours, is then evaporated in vacuo and removes solvent to a small amount of.Saturation NaHCO is added in gained remnants3Water
In solution and ethyl acetate (1:1), with saturation NaHCO3Aqueous solution washs 5 times.Then, by organic phase anhydrous Na2SO4It is dry,
It is filtered to remove Na2SO4, solvent is removed in vacuum, is washed with a small amount of ethyl acetate, filters, obtains straight chain dipeptides Fmoc-DOPA
(Acetonide)-D-Glu (OtBu)-OMe (1.2g, 75%).Fmoc-DOPA(Acetonide)-D-Glu(OtBu)-OMe
(M:C37H42N2O9)([M+Na]+:Calc.681.2783Found 681.2754).
(2) synthesis of compound Cyclo [DOPA (Acetonide)-D-Glu (OtBu)]
Straight chain dipeptides Fmoc-DOPA (Acetonide)-D-Glu (OtBu)-OMe (1mmol, 0.66g) is suspended in 20%
In piperidines/ACN solution (50ml) and stir 24 hours.Gained suspension is concentrated in vacuo and is washed 5 times with ether.By what is obtained
White solid is put into drying in vacuum oven, obtains corresponding pure products Cyclic dipeptides Cyclo [DOPA (Acetonide)-D-
Glu (OtBu)] (0.23g, 56%).
Cyclo[DOPA(Acetonide)-D-Glu(OtBu)](M:C21H28N2O6)([M+Na]+:
Calc.427.1840Found715.2645).1H NMR(400MHz,DMSO-d6) δ 8.10 (d, J=2.3Hz, 1H), 8.06-
8.01 (m, 1H), 6.70 (d, J=7.8Hz, 1H), 6.64-6.54 (m, 2H), 4.05 (qd, J1=4.2, J2=2.0Hz, 1H),
3.20 (t, J=5.2Hz, 1H), 2.98 (dd, J1=13.6, J2=4.6Hz, 1H), 2.78 (dd, J1=13.7, J2=4.8Hz,
1H),2.27–2.05(m,2H),1.78(td,J1=7.9, J2=5.2Hz, 2H), 1.60 (d, J=1.3Hz, 6H), 1.37 (s,
9H).
(3) synthesis of compound Cyclo (DOPA-D-Glu)
Cyclic dipeptides Cyclo [DOPA (Acetonide)-D-Glu (OtBu)] (1mmol, 0.41g) with protection is added
The TFA:TIS:H of 25ml2In the mixed liquor of O=(95:2.5:2.5), 1h is stirred, then precipitates to obtain solid with ice ether, from
Obtained white solid is put into drying in vacuum oven after the heart (4 DEG C, 8000rpm), obtains corresponding pure products Cyclic dipeptides
Cyclo (DOPA-D-Glu) (0.27g, 89%).
Cyclo(DOPA-D-Glu)(M:C14H16N2O6)([M-H]-:Calc.307.0936Found 307.0938).1H
NMR(400MHz,DMSO-d6,CD3COOD) δ 11.95 (s, 1H), 8.80 (s, 2H), 8.08 (d, J=2.5Hz, 1H), 7.98 (s,
1H),6.63–6.53(m,2H),6.40(dd,J1=8.0, J2=2.0Hz, 1H), 3.99 (q, J=3.9Hz, 1H), 3.08 (t, J
=5.0Hz, 1H), 2.91 (dd, J1=13.7, J2=4.3Hz, 1H), 2.68 (dd, J1=13.7, J2=4.7Hz, 1H), 2.20
(ddd,J1=16.2, J2=9.9, J3=6.1Hz, 1H), 2.08 (ddd, J1=16.2, J2=10.2, J3=5.7Hz, 1H),
1.77(qq,J1=14.2, J2=8.1, J3=6.5Hz, 2H).
Pass through the type of the Cyclic dipeptides of Fmoc-DOPA (Acetonide)-OH and H-DOPA (Acetonide)-OMe synthesis
It is summarized as follows with yield:
Table 1 passes through the Cyclic dipeptides of Fmoc-DOPA (Acetonide)-OH synthesis
Table 2 passes through the Cyclic dipeptides of H-DOPA (Acetonide)-OMe synthesis
The synthesis of straight chain dipeptides
The two kinds of synthetic method introductions of the dipeptides of straight chain containing DOPA
We use two synthetic routes, to be respectively synthesized the straight chain dipeptides containing DOPA.
Reaction route one (DOPA on the right H-AA-DOPA-OH): the side chain of proposed adoption laboratory purchase is protected with (Boc)
Amino acid and fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) form boc-protected amino acid succinimidyl carbonate
(Boc-AA-OSu), intermediate Tfa-Dopa (the Acetonide)-OMe reaction finally synthesized with us generates tert-butyl ester protection
Dipeptides.
(DOPA is in left side H-DOPA-AA-OH): using the tert-butyl ester and intermediate of bought amino acid for reaction route two
Boc-DOPA (Acetonide)-OH directly reacts the dipeptides for generating tert-butyl ester protection.
DOPA is being respectively obtained after the dipeptides of the right and left, finally in 95% trifluoroacetic acid/2.5% triisopropyl silicon
Alkane/2.5% water (95%TFA/2.5%Tis/2.5%H2O it) takes off side chain protecting group and obtains target straight chain dipeptides, Jin Erke
To carry out the research of bioactivity to it.Finally we have synthesized 19 straight chain dipeptides containing DOPA.
(1) synthetic route (reaction route one) of straight chain dipeptides H-AA-DOPA-OH
(2) synthetic route (reaction route two) of straight chain dipeptides H-DOPA-AA-OH
The 19 dipeptide structure formulas of straight chain containing DOPA synthesized at present:
The synthesis of straight chain dipeptides H-DOPA-Gly-OH, H-DOPA-Lys-OH, H-DOPA-Glu-OH
It is respectively adopted Boc-DOPA (Acetonide)-OH and glycine, the tertiary butyl ester of lysine and glutamic acid is direct
Reaction generates the dipeptidase derivant containing DOPA, and TFA removal protecting group obtains dipeptides containing DOPA.Synthetic route is as follows:
The synthesis of DOPA intermediate B oc-Dopa (Acetonide)-OH
L-DOPA (8.75g, 25mmol) is added in 200ml alkalinity LiOH solution (1.8g, 75mmol LiOH addition
To the THF/H of 200ml 3:12O solution) in 0 DEG C of reaction 2.5h.After adjusting pH to 7-8 with the HCl of 1N after the reaction was completed, then
Na is added2CO3(5.3g, 50mmol) and (Boc)2In O (6.55g, 30mmol) reaction process with TLC monitor response situation, about 5
Reaction in a hour is completed.Then pH to 3 is adjusted with the HCl of 1N, be extracted with ethyl acetate, nothing is added after washing in organic layer washing
Water magnesium sulfate water removal；Filtering, filtrate is collected and is concentrated into light residue.The product tentatively obtained is further purified, few
After measuring DCM dissolution, n-hexane to solution is added white opacity occur, adds 38 DEG C or so of a small amount of DCM water-bath to clarify to solution, be put into-
20 DEG C of refrigerator 2h recrystallizations.Obtain white powdery solids (7.96g, 84%).
Embodiment 4, the DOPA synthesized by Tfa-DOPA (Acetonide)-OMe are in right straight chain dipeptides
This reaction route is suitable for the synthesis of DOPA (H-AA-DOPA-OH) straight chain dipeptides on the right, using tertbutyloxycarbonyl
(Boc) the amino acid succinimidyl carbonate (Boc-AA-OSu) protected, the intermediate Tfa-Dopa synthesized with us
(Acetonide)-OMe reaction generates the straight chain dipeptides of contracting acetone and tertbutyloxycarbonyl protection.Finally in high concentration trifluoroacetic acid
All protecting groups are taken off under effect, obtain target straight chain dipeptides.The synthesis of DOPA (H-AA-DOPA-OH) straight chain dipeptides on the right
Route is as follows:
The synthetic route of A.H-Gly-DOPA-OH is as follows
(1) synthesis of compound Boc-Gly-OSu
Boc-Gly-OH (1.75g, 10mmol) is added in the dry round-bottomed bottle of 250mL and magnetic stir bar is added,
The dry THF of 100ml3A molecular sieve is to being completely dissolved.After to be dissolved, DCC (2.17g, 10.50mmol), HOSU is added
(1.21g, 10.50mmol) reacts 16 hours under confined conditions in room temperature.After the reaction was completed, it filters and removes insoluble white precipitate
Dicyclohexylurea (DCU) (DCU), and rinsed with THF, merging filtrate is simultaneously rotated to a small amount of stand-by.
(2) synthesis of compound Boc-Gly-DOPA (Acetonide)-OH
In the round-bottomed flask of a 250mL be added compound Tfa-Dopa (Acetonide)-OMe (3.47g,
10mmol), 120mL THF and a magnetic stir bar.Reaction flask is cooling stand-by in ice bath.Prepare 1.5mol/L LiOH
LiOH (0.72g, 30mmol) is added in the ultrapure water of 20mL by solution 20ml.Configured LiOH solution is slowly added dropwise
Into flask, start to stir.In reaction process use Silica gel 60F254 silica gel thin-layer chromatography plate contact plate, detection react into
Journey.Fully reacting after 2.5h adjusts pH to 8 with the HCl that concentration is 1mol/L.Stablize to system pH, adds anhydrous Na2CO3
The compound Boc-Gly-OSu (2.72g, 10mmol) that (2.12g, 20mmol) and the first step obtain, reacts 0.5h under ice bath
After remove ice bath, Silica gel 60F254 silica gel thin-layer chromatography plate contact plate is used in reaction process, detects extent of reaction.After 2h
Fully reacting.Then above-mentioned mixed solution is acidified to pH with the HCl of 1mol/L is about 3, is extracted with EtOAc, is then used
Twice of milli-Q water.The anhydrous MgSO of organic layer after washing4Dry 3h, after filtering filtrate again rotary evaporation to a small amount of liquid
Body.It is finally recrystallized at -20 DEG C using EtOAc/Hexane, obtains white powder crystallization.In a vacuum drying oven by it
Straight chain dipeptides Boc-Gly-DOPA (Acetonide)-OH (3.20g, 81%) of side chain protection is finally obtained after drying.
Boc-Gly-DOPA(Acetonide)-OH(M:C19H26N2O7)HRMS:([M+H-]:Calc:
393.1685Found:393.1667).
(3) it is deprotected the synthesis of straight chain dipeptides H-Gly-DOPA-OH
It takes Boc-Gly-DOPA (Acetonide)-OH (100mg, 0.25mmol) in 10ml round-bottomed flask, 3ml is added and cuts
Except 95% trifluoroacetic acid of liquid/2.5% tri isopropyl silane/2.5% water [TFA:Tis:H2O=(95:2.5:2.5)], magnetic force stirs
It mixes, reacts 2 hours at room temperature, rotary evaporation falls volatile liquid after the reaction was completed, and drains completely in a vacuum drying oven
Solvent 3h is transferred in 50ml centrifuge tube after being eventually adding 100 μm of l methanol dissolutions, is settled out immediately after 40ml ice ether is added
Centrifuge tube is finally put into centrifuge centrifugation 5min (4 DEG C, 8000rpm) by white flock precipitate, outwell obtain after supernatant it is white
Color solid precipitating is immediately placed in vacuum oven dry 4 hours, and 5ml deionized water dissolving dipeptides is added after draining solvent
It is put into -20 DEG C of refrigerators, after being frozen into solid ice completely, is put into freeze dryer freeze-drying 48h, finally obtains fluffy white solid H-
Gly-DOPA-OH (58mg, 91%).
Embodiment 5, the DOPA synthesized by Boc-DOPA (Acetonide)-OH are in left straight chain dipeptides
This reaction route is suitable for DOPA in the synthesis of the left side (H-DOPA-AA-OH) straight chain dipeptides, using normal using 20 kinds
See the tert-butyl ester and intermediate B oc- of amino acid (Gly, Ala, Leu, Ile, Val, Phe, Met, Ser, Tyr, Asp, Glu, Lys)
DOPA (Acetonide)-OH directly reacts the straight chain dipeptides for generating the tert-butyl ester and the protection of contracting acetone, then in high concentration trifluoro second
Side chain protecting group is sloughed under acid effect, obtains target straight chain dipeptides H-DOPA-AA-OH.
The synthetic route of H-DOPA-AA-OH is as follows:
The synthetic route of A.H-DOPA-Gly-OH
(1) synthesis of compound Boc-DOPA (Acetonide)-Gly-OtBu
By Boc-DOPA (Acetonide)-OH (3.38g, 10mmol) and H-Gly-OtBu.HCl (1.68g, 10mmol),
PyBOP (5.2g, 10mmol) is dissolved in the dry DCM of 100ml 3A molecular sieve, and DIEA is slowly added dropwise to after dissolving in stirring
(3.3ml, 20mmol) is reacted at room temperature 24 hours.TLC monitoring reaction process (has seen whether free ammonia in conjunction with ninhydrin detection
Base).After the reaction was completed, then vacuum concentration solvent is added 50ml and is saturated NaHCO to 50ml or so3Aqueous solution and ethyl acetate
(1:1) extraction is saturated NaHCO with equivalent3Aqueous solution washs 5 times, and deionization is washed 3 times.Then, by organic phase anhydrous slufuric acid
Magnesium removes water 3 to 4 hours, is removed by filtration anhydrous magnesium sulfate later, and rinsed with DCM.Merging filtrate rotary evaporation removes solvent, most
It is recrystallized to give target straight chain dipeptides Boc-DOPA (Acetonide)-Gly-OtBu in DCM/Hexane at -20 DEG C afterwards
(4.10g, 91%).
Boc-DOPA(Acetonide)-Gly-OtBu(M:C23H34N2O7)HRMS(ESI):([M+H]+
Calcd.451.2439,Found 451.2451).NMR spectroscopy:1H NMR(400MHz,Chloroform-d)δ
6.57 (qd, J=7.7,3.6Hz, 4H), 5.13 (d, J=8.5Hz, 1H), 4.34 (q, J=7.7Hz, 1H), 3.96-3.77 (m,
2H), 3.03-2.86 (m, 2H), 1.61 (s, 6H), 1.43 (d, J=1.7Hz, 9H), 1.37 (d, J=2.2Hz, 9H)
(2) it is deprotected the synthesis of straight chain dipeptides H-DOPA-Gly-OH
It takes Boc-DOPA (Acetonide)-Gly-OtBu (100mg, 0.22mmol) in 10ml round-bottomed flask, is added and prepares
Good 3ml cuts off liquid TFA:Tis:H2O=(95:2.5:2.5), magnetic agitation are reacted 2 hours at room temperature, are revolved after the reaction was completed
Turn to evaporate solvent, 3h drains solvent completely in vacuum oven, be eventually adding after the dissolution of 100 μm of l methanol be transferred to 50ml from
In heart pipe, 40ml ice ether is added and generates white flock precipitate immediately, is finally centrifuged 5min (4 DEG C, 8000rpm), outwells supernatant
White solid precipitating is obtained after liquid, is added 20ml ice ether and is vortexed 2 minutes, repeated washing twice, is centrifuged after outwelling supernatant,
Dry 4 hours are immediately placed in vacuum oven, and addition 5ml deionized water dissolving dipeptides is put into -20 DEG C of ice after draining solvent
In case, after being frozen into ice completely, it is put into freeze dryer freeze-drying 48h, finally obtains fluffy white solid H-DOPA-Gly-OH
(49.78mg, 89%).
H-DOPA-Gly-OH(M:C11H14N2O5)HRMS(ESI):([M+H]+Calcd.255.0975,Found
255.0976)。
Table 3, experiment synthesize DOPA straight chain dipeptides type and yield on the right
Table 4, synthesis DOPA are in left side straight chain dipeptides type and yield
Containing three peptide symthesis method of DOPA
The amine either bought using Fmoc-DOPA- (the Acetonide)-OH DOPA intermediate of laboratory preparation as raw material
The protected amino acid of base, its C-terminal is connected on resin in the form of covalent bond, and sloughs N-terminal blocking group, then according to
The conventional method of synthesis in solid state is sequentially connected amino acid, is finally cut it from resin with TFA to obtain target tripeptides.
The synthesis of embodiment 6, Fmoc-Phe-DOPA (acetonide)-Phe-OH
(1) Fmoc-L-Phe-OH is connected with resin
CTC resin (3g replaces chlorine=1mmol/g) is weighed to be placed in the synthesis in solid state pipe of a 50mL.In synthesis in solid state
Appropriate DCM wobbler swelling 20min (making to be uniformly mixed using wobbler with post-processing), vacuum water pumping is added in Guan Zhongxian
Fall DCM, 10% SOCl is added2/DCM activates 1h, and decompression filters, and DCM is added and washes 5 times.Accurately weigh 1.16g Fmoc-L-
Appropriate DMF is added in a 50mL centrifuge tube in Phe-OH (MW:386.42) (1 times of epoxy equivalent), mediates, and keeps it just molten
DIEA (3.96mL, 8eq) is added after solution, places 10min, is added in synthesis in solid state pipe and reacts 5h.After the reaction was completed, decompression is taken out
Filter, DMF are washed 5 times, and 20%MeOH/DCM is added in the resin drained, and are mixed, end socket 2h, and decompression filters, and DMF is washed 5 times, drained
Afterwards, a small amount of product is taken to carry out ninhydrin detection, ninhydrin detects (yellow).
5% piperazine/DMF is added into synthesis in solid state pipe, is uniformly mixed, after reacting 30min, DMF is washed 5 times, takes a small amount of production
Object carries out ninhydrin detection, for example navy blue, then carries out next reaction, for example light yellow, then again with 5% piperazine/DMF
Deprotection 30min illustrates that first amino acid is not successfully connected resin, needs to re-operate if light yellow not yet.
(2) Fmoc-DOPA (acetonide)-OH is connected with Fmoc-L-Phe-OH
Accurately weigh Fmoc-DOPA (acetonide)-OH (MW:459.49) (2.76g, 6mmol) (first amino acid 2
Times equivalent), BOP (2.65g, 1eq), HOBT (0.81g, 1eq) are in a 50mL centrifuge tube.Appropriate DMF is added, mediation makes it
Just it after dissolution, is added DIEA (1.98mL, 2eq), places 10min, be then added in synthesis in solid state pipe and react 2h.Wait react
Cheng Hou, decompression filter, and DMF is washed 5 times, take a small amount of product to carry out ninhydrin detection, for example navy blue, need to rejoin second
Amino acid, repetitive operation；If light yellow, illustrate that second amino acid is successfully connected resin, the reaction of next step can be started.
Appropriate 5% piperazine/DMF is added into the resin drained.30min is reacted after mixing.Decompression filters, and DMF washes 5
It is secondary, take a small amount of product to carry out ninhydrin detection, it is for example light yellow, need to rejoin 5% piperazine/DMF, repetitive operation；For example
Navy blue illustrates that second amino acid is successfully connected resin, can start the reaction of next step.
(3) Fmoc-L-Phe-OH is connected with Fmoc-DOPA (acetonide)-OH
Accurately weigh Fmoc-Phe-OH (MW:386.42) (2.32g, 6mmol) (first amino acid, 2 times of equivalents), BOP
(2.65g, 1eq), HOBT (0.81g, 1eq) are in a 50mL centrifuge tube.Appropriate DMF is added, after mediation dissolves it just,
It is added DIEA (1.98mL, 2eq), places 10min, be then added in synthesis in solid state pipe and react 2h.To which after the reaction was completed, decompression is taken out
Filter, DCM are washed 5 times, are taken a small amount of product to carry out ninhydrin detection, for example navy blue, are needed to rejoin third amino acid, are repeated
Operation；If light yellow, illustrate that third amino acid is successfully connected resin, the reaction of next step can be started.
Appropriate 2%TFA/DCM lysate is added into synthesis in solid state pipe.Cracking 3 times, the time of cracking is successively about
30min, 60min, 90min filter resin, and collecting merging, filtrate is added ice ether and mixes, sink in 50mL centrifuge tube three times
Drop, supercentrifuge 8000rpm are centrifuged 15min, and three times, argon gas dries up after removing impurity, dried in vacuum oven for centrifugation
At night, taking a small amount of sample HPLC, sample introduction is analyzed.Elution process is as shown in table 5.HPLC and HR-MS analysis: HPLC:RT=19min,
HR-MS(ESI):C45H43N3O8, MH+Calcd.754.3050, Found 754.3127.
The elution process of table 5 Fmoc-Phe-DOPA (acetonide)-Phe-OH
Anti- degradation capability introduction in the liver homogenate of oligopeptides containing DOPA
Human small intestine's wall not only has the channel for absorbing unimolecule amino acid, and there are also the specific channels for directly absorbing oligopeptides, respectively
Kind experiment confirms that dipeptides containing DOPA is easier to enter blood circulation by intestinal wall than DOPA, and the half-life period in blood plasma is longer, has
Stronger anti-Parkinson's disease activity, while also there is certain slow release effect.Relative to straight chain dipeptides, Cyclic dipeptides structure is generally come
Say more stable, it should be more stable in human gastrointestinal tract, blood circulation and liver, and half-life period is longer, may have stronger
Anti-Parkinson's disease activity.
The liver homogenate degradation experiment of the dipeptides of straight chain containing DOPA:
(1) it pre-processes
Rat pretreatment: purchased SD rat (healthy male SD rat (180g-220g)) is raised at 20~25 DEG C of temperature, phase
To humidity 40%~60%, under the conditions of natural lighting, 4, every cage conventinal breeding 2 weeks, replaces padding every other day.First 12 hours of experiment
Fasting, free water.Dissection reagent used and dissection apparatus are first placed in 4 DEG C of pre-coolings.
Liver homogenate preparation: it after rat is anesthetized with ether, opens abdomen and takes out liver.The liver of taking-up is at 4 DEG C or less on ice with life
Reason salt water is rinsed to grey, with filter paper suck dry moisture and is weighed.Then in shredding in culture dish, the liver that 2.0g is shredded is accurately weighed
It is dirty to be homogenized on refiner in 10ml centrifuge tube, it then draws 4mL PBS liquid and is added thereto, be prepared into 50% liver of mass fraction
Homogenate, then be fully ground with tissue homogenizer, after supercentrifuge is centrifuged 10min (8000r/min, 4 DEG C), draw supernatant
Liquid finally obtains hepatic tissue liquid.The liver homogenate liquid prepared is saved to -80 DEG C of ultra low temperature freezers, it is spare.
(2) liver homogenate degradation experiment
64 μm of ol oligopeptides containing DOPA are accurately weighed in a 2mL centrifuge tube, are added in the PBS solution of 1mL, vortex oscillation
It is configured to the sample solution that concentration is 64mM.Then it in 2mL centrifuge tube, takes 500 μ L of sample liquid and 50% liver homogenate liquid is added
500 μ L are uniformly mixed, and are placed in 37 DEG C of constant-temperature incubations, start reaction and timing is 0min.A 2mL centrifuge tube separately is taken, takes sample
50 μ L of liquid and be added 50 μ L of PBS liquid be uniformly mixed, as 0min compare.
(3) HPLC is analyzed:
HPLC condition: sampling sample uses the dissolution of chromatographic grade acetonitrile to be configured to concentration as 1mg/ml, and mobile phase is pure using (A)
Acetonitrile；Mobile phase (B) pure water/10mM NH4Ac (pH=4)；Type of elution: by mobile phase 95%A to mobile phase 50%B gradient
Elute 20min, flow velocity 1ml/min, sample volume 20 μ L, 25 DEG C of column temperature, Detection wavelength 285nm；Chromatographic column: Waters-Xbridge
Amide hilic column,250mm×4.6mm,5μm.
Preliminary experiment: the difference due to not knowing the different dipeptides of straight chain containing DOPA degradation times in liver homogenate, so each
When sample first set reaction, corresponding sample interval should be adjusted according to HPLC peak area variation tendency.Sampling: reaction one
40 μ L are sampled according to the determined time interval of preliminary experiment after the section time, 360 μ L anhydrous methanol vortex oscillations, supercentrifuge is added
Be centrifuged 3min (8000r/min, 4 DEG C), after the completion of centrifugation with 1ml syringe take supernatant by nylon micro porous filter membrane (organic film,
0.22 μm) filtering, filtrate progress HPLC analysis.Sample introduction is analyzed: according to preliminary result, selecting sample interval, chooses
About 8 sample points are handled according to standard operation after every sub-sampling, and sample introduction is analyzed by HPLC, according to the peak area ratio of HPLC feedback into
Row adjustment, it is final to choose 5 sample points that sufficiently show degradation trend.Then by the integrating peak areas of different time points,
Function Fitting obtains the degradation half life t of the different oligopeptides containing DOPA1/2(min)。
The half-life period t1/2 (min) that L-DOPA straight chain dipeptides is degraded in rat liver homogenate
The peak area S that HPLC is obtained after the sample feeding normally measured is taken, and by it using the time as the natural logrithm of variable
Curve is made, result substantially conforms to level-one curve law.It is as follows according to fitting function Calculation results, straight chain containing DOPA two
The half-life period of peptide, half-life difference was larger under this experiment condition.Wherein Asp modifies the half-life period longest of the straight chain dipeptides of DOPA,
Long half time reaches 174min；The half-life period that Met modifies the straight chain dipeptides of DOPA is most short, and half-life period is only 16min.
The liver homogenate degradation half life of 6 dipeptides of straight chain containing DOPA of table
Note:1Amino acid indicates the amino acid residue in H-DOPA-AA-OH in table
The half-life period t1/2 (min) that Cyclic dipeptides containing L-DOPA are degraded in rat liver homogenate
The half-life period of the Cyclic dipeptides containing DOPA, half-life difference was larger under this experiment condition.Wherein Lys, DOPA, Gln, Ser
The half-life period for modifying the Cyclic dipeptides of DOPA is shorter, and Lys half-life period is 502min；Trp, Glu, D-Glu, Gly, Ala modify DOPA
Cyclic dipeptides half-life period it is longer, Ala half-life period longest, reach 3419min.Shown in table specific as follows:
The liver homogenate degradation half life of 7 Cyclic dipeptides containing DOPA of table
Note:2Amino acid indicates the amino acid residue in cyclo (DOPA-AA) in table
The dipeptides containing DOPA that the method for the present invention is prepared, especially Cyclic dipeptides have in liver homogenate to be stablized well
Property, anti-degradation capability is strong, is expected to be developed into bioavilability height, long-acting Antiparkison Drugs, simultaneously as it is given birth to
Object availability greatly improves, and is expected to that the side effect of L-DOPA treatment Parkinson's disease is greatly reduced.
Claims (10)
1. a kind of liquid-phase synthesis process of dipeptides containing DOPA, synthetic route are as follows:
In formula, R1And R2In one be protection DOPA side chain, preferred protecting group be contracting acetone, another for protect or not
The amino acid side chain of protection；
R5For amino protecting group, R6For carboxyl-protecting group；Synthetic method includes the following steps:
It willIt is dissolved in organic solvent A, condensing agent is added, liquid phase condensations react to obtain straight
Chain dipeptides R5-AA1-AA2-OR6；
By straight chain dipeptides R5-AA1-AA2-OR6Deprotection obtains straight chain dipeptides；Or by straight chain dipeptides R5-AA1-AA2-OR6It is dissolved in
It is reacted in alkalinity deprotection liquid, liquid phase reactor sloughs amino protecting group, the Cyclic dipeptides containing DOPA protected
2. liquid-phase synthesis process according to claim 1, it is characterised in that: it is more to further use acid deprotection liquid removing
Bar Side chain protective group, obtains the Cyclic dipeptides containing DOPA containing free catechol group.
3. liquid-phase synthesis process according to claim 1, it is characterised in that: R1、R2Be independently selected from L-type or D type Gly, Ala,
Val, Leu, Ile, Phe, Trp, Tyr, Asp, Asn, Glu, Lys, Gln, Met, Ser, Thr, Cys, Pro, His, Arg or DOPA
Amino acid side chain.
4. described in any item liquid-phase synthesis process according to claim 1~3, it is characterised in that: R5Selected from Fmoc, Boc；R6Choosing
From methyl, tert-butyl.
5. described in any item liquid-phase synthesis process according to claim 1~3, it is characterised in that: organic solvent A, alkaline remove-insurance
The solvent of shield liquid is independently selected from acetonitrile, N,N-dimethylformamide (DMF), methylene chloride (DCM), ethyl acetate (EA) at least one
Kind.
6. described in any item liquid-phase synthesis process according to claim 1~3, it is characterised in that: acidity deprotection liquid, which is selected from, to be contained
The solution of TFA, HCl.
7. described in any item liquid-phase synthesis process according to claim 1~3, it is characterised in that: the alkali in alkalinity deprotection liquid
Property ingredient be selected from piperidines, piperazine.
8. liquid-phase synthesis process according to claim 7, it is characterised in that: quality percentage of the piperidines in alkaline solution contains
Amount is 5~60%, preferably 15~50%.
9. new intermediate product or final product that any one of claim 1~8 liquid-phase synthesis process synthesizes.
10. the dipeptides containing DOPA prepare L-DOPA prodrug, anti-Parkinson's disease sustained release prodrug in application, in which:
The structural formula of Cyclic dipeptides containing DOPA are as follows:
The structural formula of straight dipeptides containing DOPA are as follows:
In formula, R1For L-type or the side chain of D type amino acid；
Preferably, the middle R of the straight dipeptides containing DOPA1For L-type or the side chain of D type Gly, Leu, Val, Phe, Ile, Tyr or Asp；
Preferably, the middle R of the Cyclic dipeptides containing DOPA1For L-type or the side of D type Gly2, Ala, Trp, Lys, Glu, Gln, Ser, DOPA
Chain.
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| CN111153885A (en) * | 2019-02-18 | 2020-05-15 | 海南大学 | Synthesis method of intermediate of dopa oligopeptide, application, composition and preparation thereof |
| WO2022191338A1 (en) * | 2021-03-10 | 2022-09-15 | Mitsubishi Tanabe Pharma Corporation | Levodopa-prodrugs and carbidopa-prodrugs for use in the treatment of parkinson's disease |
| WO2022190100A1 (en) * | 2021-03-10 | 2022-09-15 | Neuroderm, Ltd. | Stabilized liquid compositions comprising a levodopa-tyrosine conjugate and uses thereof |
| US11844754B2 (en) | 2020-11-17 | 2023-12-19 | Neuroderm, Ltd. | Methods for treatment of Parkinson's disease |
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| CN111153885A (en) * | 2019-02-18 | 2020-05-15 | 海南大学 | Synthesis method of intermediate of dopa oligopeptide, application, composition and preparation thereof |
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| WO2022190100A1 (en) * | 2021-03-10 | 2022-09-15 | Neuroderm, Ltd. | Stabilized liquid compositions comprising a levodopa-tyrosine conjugate and uses thereof |
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