CN106631957A - Antitumor compound targeting FAP-alpha enzyme and preparation method and application thereof - Google Patents
Antitumor compound targeting FAP-alpha enzyme and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an antitumor compound targeting an FAP-alpha enzyme. The antitumor compound is named Z-GP-procarbazine; a dipeptide part (Z-GP) in the compound can be removed by specific hydrolysis of the FAP-alpha enzyme to release procarbazine. The Z-GP-procarbazine can significantly lower the toxicity and in-vivo toxicity of normal cells, can be free from the dipeptide part (Z-GP) in vivo and in vitro through specific hydrolysis of the FAP-alpha enzyme to release an enzymolysis product, and can significantly inhibit the growth of tumors in nude mice bearing tumors and lower the toxicity to non-target organs, thus reducing the deficiency of severe toxic and side effects of procarbazine as an anticancer drug. The invention provides a tumor targeting drug based on FAP-alpha. In addition, the invention discloses a preparation method of the Z-GP-procarbazine. The method has the characteristics of mild reaction conditions, simple experiment steps, high yield, high purity of a product, economical efficiency, practicability and the like. In addition, the invention discloses use of the Z-GP-procarbazine, a derivative of the Z-GP-procarbazine and a physiologically-acceptable salt of the Z-GP-procarbazine in preparation of antitumor drugs.
Description
Technical field
The present invention relates to a kind of compound and preparation method thereof, especially a kind of antitumorization of targeting FAP-alpha enzymes
Compound and preparation method and application, belongs to field of antineoplastic medicaments.
Background technology
Currently, the clinical chemotherapy medicine of tumour is mainly cytotoxic drug, although they possess stronger antitumor work
Property, lack selective yet with this kind of chemotherapeutics, often lead to serious toxic and side effect.So, realize cytotoxicity medicine
The targeting Delivery of thing has significant meaning to the bad reaction for reducing chemotherapy of tumors.
Procarbazine (procarbazine, Pcb) is clinically mainly used in treating Hodgkin's disease, pernicious swollen for other
Knurl such as malignant lymphoma, lung cancer, Huppert's disease etc. also have certain curative effect.Nineteen eighty-two the medicine hydrochloride with oral capsule
Formulation is ratified, in U.S.'s listing, to be mainly used in the treatment of malignant lymphoma by FDA.Lack anticancer yet with this kind of chemotherapeutics
It is selective, often lead to larger toxic and side effect, the especially poisoning effect to reproduction cell and bone marrow suppression.It is well known that bone
Marrow suppresses that leucocyte, decrease of platelet can be caused, and has hemorrhagic tendency, can also cause anaemia.These toxic and side effects limit it and are facing
Application on bed.
It is to carry out structural modification to medicine to solve one of effective way of antineoplastic toxic and side effect, makes precursor
Medicine, using the difference on molecular biology between tumour cell and normal cell, selectively acting is in the base of tumour target cell
Cause, enzyme, information conduct factors etc., so as to reach the purpose of targeted therapy.Substantial amounts of research in recent years shows that fibroblast swashs
Living protein alpha (FAP-alpha) is the mesenchyma stroma of tumors antigen molecule expressed by tumour associated fibroblast cell-specific height, right
The generation development of tumour has important facilitation.FAP-alpha has specific endopeptidase activity, can be selective
The glycyl proline dipeptides sequence of ground hydrolyzing N-endcapped, such as carries the substrate of Z-Gly-Pro (Z-GP) dipeptides.Therefore,
Fibroblast activation protein alpha (FAP-alpha) is selected to be the effective plan for improving oncotherapy composite index as target
One of slightly.
The content of the invention
It is an object of the invention to provide a kind of antitumoral compounds of targeting FAP-alpha enzymes, the compound energy quilt
FAP-alpha enzyme spcificitys hydrolysis excision dipeptide moieties (Z-GP), discharge procarbazine, are named as Z-GP- procarbazine (Z-
GP-Pcb), the Z-GP-Pcb can overcome the shortcomings of directly using procarbazine as during cancer therapy drug with serious toxic and side effect,
The toxicity and toxicity in vivo of normal cell can be significantly reduced, the growth of tumor bearing nude mice in-vivo tumour can be significantly inhibited and reduced right
Non-target organ toxicity, is expected to develop into new antineoplastic.
For achieving the above object, the technical scheme taken of the present invention is：A kind of antitumor chemical combination of targeting FAP-alpha enzymes
Thing, its structure is shown in formula I：
Or shown in Formulas I compound isomer, its structure is as shown in Formula II：
The Compound nomenclature is Z-GP- procarbazines (Z-GP-Pcb), and Formulas I and Formula II are isomer, Liang Zhe
There is same effect in drug metabolism, medicinal usage, be mesenchyma stroma of tumors fibroblast activator protein enzyme alpha (FAP-
Alpha specific for hydrolysis substrate), its molecular weight is 509.26D α.
In addition, the present invention provides a kind of preparation method of the compound Z-GP- procarbazines, methods described includes following
Step：
(1) by procarbazine, benzyloxycarbonyl group glycyl proline (Z-GP-OH) and condensation reagent, stir at 0 DEG C～30 DEG C
Mix and react, obtain mixture；
(2) after completion of the reaction, saturated salt solution is added in step (1) gained mixture, is quenched, is separated, is purified,
Obtain final product compound Z-GP- procarbazines.
As the preferred embodiment of the preparation method of compound of the present invention, the condensation reagent in the step (1)
For I-hydroxybenzotriazole and N, N- diisopropylethylamine and 2- (7- aoxidizes BTA)-N, N, N', N'- tetramethylurea six
Fluorophosphoric acid ester, ethyl chloroformate, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, N, N '-diisopropyl carbon
Diimine, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- chlorine N, in N, 2- trimethacrylate amine at least
A kind of mixture.
As the most preferred embodiment of the preparation method of compound of the present invention, the condensation examination in the step (1)
Agent is 2- (7- aoxidizes BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (HATU), I-hydroxybenzotriazole
(HOBT) and N, N- diisopropylethylamine (DIPEA) mixture.
As the preferred embodiment of the preparation method of compound of the present invention, procarbazine, benzyl in the step (1)
The mol ratio of oxygen carbonyl glycyl proline and condensation reagent is 1:(1～3.0):(1.05～3.0).
As the preferred embodiment of the preparation method of compound of the present invention, the salt in the step (2) be NaCl,
KCl、NH4Cl、(NH4)2SO4In at least one.
As the preferred embodiment of the preparation method of compound of the present invention, the procarbazine in the step (1)
Synthetic route be：
Specifically adopt and be prepared from the following method：
A p-methylbenzoic acid is dissolved in organic reagent by (), add excessive thionyl chloride, in 60-80 DEG C of oil bath heating backflow
1-3h, revolving removes excessive thionyl chloride, obtains to methyl benzoyl chloride, and organic examination then will be redissolved in methyl benzoyl chloride
In agent, isopropylamine is slowly added to, at 30 DEG C, charging is finished and continues that reaction 2-4h, Ran Housheng is stirred at room temperature control temperature
Temperature continues to react 20-40min to 40 DEG C, and reaction is isolated and purified after terminating, and obtains N- isopropyls to methyl benzamide；
B () will be dissolved in organic reagent obtained by step (a) to methyl benzoyl chloride, be slowly added to ammonium ceric nitrate suspension,
Charging is finished, and is placed in 90-100 DEG C of oil bath pan and is reacted 20-30h, and reaction is isolated and purified after terminating, and obtains 4- formoxyl-N- isopropyls
Yl-benzamide；
C N- isopropyls obtained by step (b) are dissolved in organic reagent by () to methyl benzamide and methylhydrazinium sulphate, plus
Enter triethylamine, reaction unit is placed in 50-70 DEG C of oil bath pan and reacts 5-7h, be spin-dried for solvent, mixture is redissolved in organic
In reagent, sodium cyanoborohydride is slowly added in 0 DEG C of cryostat, charging is finished, and reaction system is gradually increased to room temperature,
Overnight, reaction is isolated and purified after terminating, and obtains final product procarbazine for reaction.
As the preferred embodiment of the preparation method of compound of the present invention, the synthesis of the Z-GP- procarbazines
Route is：
As the preferred embodiment of the preparation method of compound of the present invention, the organic solvent in the step (a)
For dry dichloromethane or chloroform, the p-methylbenzoic acid is 1.0 with the mol ratio of thionyl chloride:(5.0～100), institute
It is 1 that p-methylbenzoic acid is stated with the mol ratio of isopropylamine:(5.0～10).
As the preferred embodiment of the preparation method of compound of the present invention, the organic solvent in the step (b)
For salpeter solution or acetum, the concentration of salpeter solution is 3.0-5.0mmol/L, the N- in the ammonium ceric nitrate suspension
Isopropyl is 1 to the molar feed ratio of methyl benzamide and cerous nitrate:(3.0～5.0).
As the preferred embodiment of the preparation method of compound of the present invention, the organic solvent in the step (c)
For absolute ethyl alcohol or DMF, the 4- formoxyls-N- isopropylbenzamides are 1 with the mol ratio of methylhydrazinium sulphate:(3.0～
5.0)。
In addition, the present invention provides a kind of antitumoral compounds of targeting FAP-alpha enzymes, the compound is described above
The derivative or physiologically acceptable salt of compound Z-GP- procarbazines.Z-GP- procarbazines or Z-GP- procarbazines are given birth to
Acceptable salt can exist in medicinal in free state form in reason.
Used as the preferred embodiment of compound described above of the invention, the compound Z-GP- procarbazines are physiologically
Acceptable salt is tartrate, sulfate, hydrochloride.Preferably, the Z-GP- procarbazines physiologically acceptable salt
Preparation method is comprised the following steps：Z-GP- procarbazines are dissolved in the organic solvent of the acid containing 1.05～3.0 moles (HA),
The stirring reaction 3～20 hours at a temperature of -10 DEG C～40 DEG C, separates solid chemical compound, washs, then solid chemical compound is again molten
Yu Shuizhong, freeze-drying obtains final product the physiologically acceptable salt of compound Z-GP- procarbazines.Preferably, it is described above organic molten
It is 1 that agent is volume ratio:1 methyl alcohol and the mixed solution of dichloromethane.
In addition, the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes compound Z-GP- third described above
The derivative of carbonohydrazides and/or Z-GP- procarbazines and/or or Z-GP- procarbazines in physiologically acceptable salt.
In addition, a kind of compound described above of present invention offer or described pharmaceutical composition are in antineoplastic is prepared
Using.
Used as the preferred embodiment of application of the present invention, the compound or pharmaceutical composition are mesenchyma stroma of tumors into fibre
The specific for hydrolysis substrate of cell-stimulating albumen enzyme α.
Used as the preferred embodiment of application of the present invention, the tumour is malignant lymphoma, lung cancer, liver cancer or multiple
Property myeloma.
The beneficial effects of the present invention is：(1) Z-GP- procarbazines of the present invention can significantly reduce the poison of normal cell
Property and toxicity in vivo, in vivo and in vitro can by FAP-alpha enzyme spcificitys hydrolysis excision dipeptide moieties (Z-GP) discharge hydrazinolysis produce
Thing；(2) the Z-GP- procarbazines can significantly inhibit the growth of tumor bearing nude mice in-vivo tumour and reduce to non-target organ poison
Property；(3) preparation method of Z-GP- procarbazines of the present invention have that reaction condition is gentle, experimental procedure simple, high income,
Product purity is high, it is economical and practical the features such as.
Description of the drawings
Fig. 1 is enzymolysis effect figures of the FAP-alpha to compound Z-GP- procarbazines of the present invention；
Fig. 2 is that Pcb and Z-GP-Pcb affect comparison diagram to the cytotoxic effect of NCI-H460, wherein, with blank group phase
Than * P<0.05, * * P<0.01；
Fig. 3 is the cytotoxicity work that FAP-alpha+Z-GP-Pcb coprocessing and Pcb process is respectively adopted to NCI-H460
With impact comparison diagram, compared with blank group, * P<0.05, * * P<0.01；
Fig. 4 is the comparison diagram of Pcb and Z-GP-Pcb to mouse testis weight, wherein, compared with blank group,##P<
0.01, compared with Pcb groups, * * P<0.01；
Fig. 5 is the comparison diagram that Pcb and Z-GP-Pcb affect on mouse sperm quantity, wherein, compared with blank group,#P<
0.05, compared with Pcb groups, * P<0.05；
Fig. 6 is the drug distribution comparison diagram of Pcb and Z-GP-Pcb in tumour and testis, wherein (A) be medicine in tumour
The accumulation situation of tissue, (B) is accumulation situation of the medicine in testis tissue；
Fig. 7 is the comparison diagram that Pcb and Z-GP-Pcb affect on H22 liver cancer tumor-bearing mices gross tumor volume size.
Specific embodiment
It is right below in conjunction with the accompanying drawings and the specific embodiments to better illustrate the object, technical solutions and advantages of the present invention
The present invention is described further.
Embodiment 1
A kind of embodiment of the preparation method of Z-GP- procarbazines of the present invention, the preparation method includes following step
Suddenly：
1.1.1 preparation of the N- isopropyls to methyl benzamide
Paratolunitrile 273mg (2.0mmol) is weighed in the round-bottomed flask of 25mL, 5.0mL (68.8mmol) two is added
Chlorine sulfoxide (excess), in 78 DEG C of oil bath heating backflow 2h；Revolving removes excessive thionyl chloride, obtains to methyl benzamide；Will
During the dichloromethane (DCM) of 2.0mL dryings is redissolved in methyl benzamide；Separately take 1.0mL (11.7mmol) isopropylamine molten
In the DCM that 2.0mL is dried, A liquid is obtained；Control temperature is gradually added to A drops in reaction system at 30 DEG C, and charging is finished
Continue to stir 30min, stirring reaction 3h under room temperature；40 DEG C are warming up to afterwards, continue to react 30min, stop heating；By reactant liquor
In being poured into 50mL frozen water, with dichloromethane-ether (volume ratio 1: 5) extract 4 times, merge organic phase, successively with water, watery hydrochloric acid,
Water, diluted sodium hydroxide solution, water washing, anhydrous magnesium sulfate is dried；Revolving removes solvent, obtains compound as white solid 289.8mg,
Yield 88.9%.
Jing analysis detections, gained compound as white solid1H NMR(300MHz,CDCl3)δ:7.67 (d, J=3.0Hz,
1H), 7.64 (d, J=3.0Hz, 1H), 7.21 (d, J=3.0Hz, 1H), 7.19 (d, J=3.0Hz, 1H), 4.22～4.33 (m,
1H),2.38(s,3H),1.26(s,3H),1.24(s,3H)；13C NMR(75MHz,CDCl3)δ:166.6,141.5,132.1,
129.1,129.1,126.8,126.8,41.7,22.8,21.3,21.3.ESI-MS m/z:178.2[M+H]+,200.3[M+
Na]+.Data above proof gained white solid product is N- isopropyls to methyl benzamide.
1.1.2 the preparation of 4- formoxyls-N- isopropylbenzamides
Take 5mL double distilled waters to be well mixed with 6mL red fuming nitric acid (RFNA)s, the salpeter solution for making about 3.5mol/L is standby；Weigh N-
Isopropyl in pressure pipe, adds 2mL 3.5mol/L salpeter solutions, ultrasound shake to methyl benzamide 177mg (1.0mmol)
Swing dissolving；Ammonium ceric nitrate 4.4g (4.0mmol) is taken in cillin bottle, 8.0mL 3.5mol/L salpeter solutions are added, 5min is stirred,
Obtain final product the nitric acid suspension of ammonium ceric nitrate；Take the ammonium ceric nitrate suspension for stirring dropwise to be added drop-wise in pressure pipe, fed
Finish, be placed in 100 DEG C of oil bath pans and react 24h；Reaction terminates, and reactant liquor is cooled down, and is subsequently added 100mL saturations NaCl water-soluble
Liquid, with DCM (3 × 100mL) is extracted, and merges organic phase, adds anhydrous sodium sulfate drying, is filtered, and is spin-dried for organic solvent and is obtained micro- Huang
Color pressed powder, with silica gel chromatography (ethyl acetate-light petrol, volume ratio 1:5) white solid 136.2mg, yield, are obtained
71.2%.
Jing analysis detections, gained compound as white solid1H NMR(300MHz,CD3OD)δ:10.03(s,1H),7.95
(d, J=9.0Hz, 2H), 7.58 (d, J=9.0Hz, 2H), 4.10～4.30 (m, 1H), 1.26 (d, J=6.0Hz, 3H), 1.24
(d, J=6.0Hz, 3H)；13C NMR(75MHz,CDCl3)δ:193.5,168.4,130.6,129.0,128.1(2),127.2
(2),43.4,22.5(2)；ESI-MS(m/z):192.3[M+H]+,224.5[M+Na]+.Data above proves gained white solid
Product is 4- formoxyl-N- isopropylbenzamides.
1.1.3 the preparation of procarbazine (procarbazine)
Weigh 191mg (1.0mmol) 4- formoxyl-N- isopropylbenzamides and 505mg (3.5mmol) methyl hydrazine sulfuric acid
Salt adds absolute ethyl alcohol 20.0mL, stirring and dissolving after stirring 20min, to add triethylamine 1.0mL in 250mL reaction bulbs；Will
Reaction unit is placed in 60 DEG C of oil bath pans and reacts 6h, and reaction terminates, and is spin-dried for solvent, and the DMF for adding 10.0mL again dissolves it,
Sodium cyanoborohydride 126mg (2.0mmol) is slowly added in 0 DEG C of cryostat, charging is finished, and reaction system gradually rises
To room temperature, reaction is overnight；After reaction terminates, add water and reaction is quenched；The saturation NaCl aqueous solution of 200.0mL is added, 200.0mL is used
Ethyl acetate extract 5 times, merge organic phase, add anhydrous sodium sulfate drying, filter, merge organic phase, be spin-dried for obtaining yellow oil
Shape liquid；Silica gel column chromatography separates (ethyl acetate-light petrol, volume ratio 1:3) procarbazine 161.9mg, yield 74%, are obtained.
Jing analysis detections, gained procarbazine1H NMR(300MHz,CDCl3)δ:7.47 (d, J=7.0Hz, 1H),
7.43 (d, J=7.0Hz, 1H), 7.31 (d, J=7.0Hz, 1H), 7.24 (d, J=7.0Hz, 1H), 3.88-3.91 (m, 1H),
3.93 (s, 2H), 2.47 (s, 3H), 1.25 (d, J=6.0Hz, 3H), 1.23 (d, J=6.0Hz, 3H)；13C NMR(75MHz,
CDCl3)δ:166.2,144.4,130.1,125.8,125.8,125.7,125.7,55.2,40.4,33.7,22.3,22.3；
ESI-MS(m/z):222.3[M+H]+,244.3[M+Na]+.Data above proves that products therefrom is procarbazine really
(procarbazine)。
1.1.4 the preparation of Z-GP- procarbazines (Z-GP-Pcb)
612mg (2mmol) Z-GP-OH is weighed, 283mg (2.1mmol) HOBt and 798mg (2.1mmol) HATU are dissolved in
10mL is dried DCM, in being poured into 50mL round-bottomed flasks, adds diisopropylethylamine (DIPEA) 0.3mL, and reaction bulb is placed in
Stir-activating 30min in ice bath；Separately weigh 442mg (2.0mmol) procarbazine and be dissolved in 5mL and be dried DCM, be poured into cillin bottle
In, DIPEA 0.5mL are added, after reaction 30min, solution is slowly dropped in the reaction bulb of activation, subsequently reaction bulb is moved
To room temperature reaction 3-4h；After completion of the reaction plus saturation NaCl solution is quenched, it is multiple with DCM extractions, merge organic phase, use successively
Water, the washing of saturation NaCl solution, anhydrous Na2SO4Remove solvent after drying under reduced pressure；Gained mixture Jing silica gel column chromatographies (chloroform-
Methyl alcohol, volume ratio 10:And RP-HPLC isolates and purifies that (eluent is MeOH 1):Water=50:50, V/V), obtain faint yellow sticky
Shape liquid 690mg, yield 65%.
Jing analysis detections, the faint yellow glutinous thick liquid of gained1H NMR(300MHz,CD3OD)δ:7.83～7.78 (m,
2H), 7.52～7.47 (m, 2H), 7.38～7.30 (m, 5H), 5.10 (d, J=6.6Hz, 2H), 4.29～3.96 (m, 5H),
3.57 (m, 2H), 3.22 (s, 1H), 3.17 (s, 2H), 2.05 (m, 3H), 1.87～1.66 (m, 2H), 1.26 (d, J=6.7Hz,
6H)；13C NMR(75MHz,CD3OD)δ:173.58,166.38,165.55,156.31,140.79,137.39,134.46,
129.11,128.77(2),128.31,127.75,127.71,127.48,127.27(2),65.88,65.84,56.59,
52.32,48.91,45.92,41.43,29.76,24.29,21.89(2)；ESI-MS(m/z):532.5[M+Na]+, above number
It is demonstrated that the faint yellow sticky shape product liquid of gained is really Z-GP- procarbazines (Z-GP-Pcb), its structure is shown in formula I：
Or shown in Formulas I compound isomer, its structure is as shown in Formula II：
The investigation experiment of the Z-GP-Pcb targeted-release characteristics of embodiment 2
Experimental technique：HPLC chromatogram condition is as follows：High performance liquid chromatograph Agilent 1200；Chromatographic column Cosmosil
C18 reverse-phase chromatographic columns (4.6 × 250mm, 5 μm)；Mobile phase (0min, 55% methyl alcohol and 45% water (ammonium formate containing 2mM)；
10min, 65% methyl alcohol and 35% water (ammonium formate containing 2mM)；15min, 75% methyl alcohol and 25% water (ammonium formate containing 2mM)；
30min, 85% methyl alcohol and 15% water (ammonium formate containing 2mM)；40min, 85% methyl alcohol and 15% water (ammonium formate containing 2mM)；Flow velocity
1mL/min；Detection wavelength：254nm；The μ L of sample size 2.
Set up Z-GP- procarbazine examination criteria curve methods as follows：Z-GP- procarbazines are dissolved in into enzymolysis buffer solution
(50mM Tris-HCl, 1.0M NaCl, pH 7.4), arrange 5 concentration gradients be respectively 0.012725,0.006363,
0.003181st, 0.001591,0.000795 μ g/ml, with peak area as ordinate, drug concentration is that abscissa draws standard song
Line, this experiment is repeated 3 times, and the Z-GP- procarbazines of final concentration of 30mM are incubated in the rhFAP- containing 2 μ g/ml and 5 μ g/ml
In the buffer solution of alpha, reaction temperature is 37 DEG C, in incubation 0,4,8,12,16,24h time points, takes the inspection of supernatant HPLC methods
Survey free procarbazine.
Experimental result：Enzymolysis test result indicate that, FAP-alpha can be catalyzed Z-GP-Pcb hydrolysis release Pcb, and its digest
Efficiency is proportionate (Fig. 1) with enzyme concentration.
The investigation experiment of cytotoxicity change before and after the Z-GP-Pcb of embodiment 3 enzymolysis
Experimental technique：By Z-GP-Pcb and Pcb respectively in the range of 0.1-50 μM of drug concentration to NCI-H460 cells
Strain (being purchased from Chinese Academy of Sciences's Shanghai cell bank) processes 48h, and using mtt assay both cytotoxicities are compared；By Z-GP-Pcb with
FAP-alpha is incubated altogether, takes incubation supernatant and processes NCI-H460 cell line 48h, using mtt assay analysis FAP-alpha enzymolysis
Impact of the effect to Z-GP-Pcb cytotoxicities, whether can to recover its potential for Z-GP-Pcb under FAP-alpha enzymolysis
Cytotoxicity.
Experimental result：Cytotoxicity experiment result shows that Z-GP-Pcb is significantly lower than Pcb to the cytotoxicity of NCI-H460
(Fig. 2)；Under FAP-alpha enzymolysis, Z-GP-Pcb recovers its CDCC (Fig. 3).
The Z-GP-Pcb Sperm toxicity research experiments of embodiment 4
Experimental technique：18-22g male mouse of kunming 60, is randomly divided into 5 groups, respectively lumbar injection 75mM Pcb/Z-
GP-Pcb and 150mM Pcb/Z-GP-Pcb, after 18 days left testes are taken, and are weighed (g), add physiological saline to prepare sperm suspension,
The supernatant sperm suspension that absorption is prepared is moved in another centrifuge tube, and being placed in 60～80 DEG C of water-bath makes dead sperm；
Take suspension 1 to drip, instill cell counting count board, count the sperm count in suspension, sperm count=suspension sperm count × extension rate.
Experimental result：Knowable in Tables 1 and 2, compare with control group, the procarbazine difference single of 75mM and 150mM
After injection 18 days, testicular weight reduces respectively 29.5% and 55.4%；Sperm count significantly reduces respectively 42.2% and 52.7%, poor
It is different statistically significant；And Z-GP-Pcb 75mM and 150mM distinguish single injection after 18 days, compared with control group, testicular weight
It is almost unchanged, sperm quantity reduce it is less obvious, respectively 33.4% and 35.3%, Z-GP-Pcb respectively with matched doses third
Carbonohydrazides compares, and testicular weight and sperm quantity substantially increase, and has significant difference (Fig. 4 and Fig. 5).
Impacts of the Z-GP-Pcb of table 1 to mouse testis weight
Note：Compared with blank group,##P<0.01, compared with Pcb groups, * * p<0.01
The Z-GP-Pcb of table 2 affects on mouse sperm quantity
Note：Compared with blank group,#P<0.05, compared with Pcb groups, * P<0.05
The Z-GP-Pcb Targeting distributions characteristic of embodiment 5 investigates experiment
Experimental technique：KM mouse 150 are taken, it is (thin purchased from Chinese Academy of Medical Sciences's tumour that H22 knurl strains are passed in aseptic absorption
Born of the same parents storehouse) and well-grown mouse ascites, with normal saline dilution into 1*107Cells/mL, mouse is inoculated in per mouse 0.2mL right
Side armpit is subcutaneous to set up bearing mouse model；Model mice (n=10) disposable vein is given 10mg/kg Z-GP-Pcb or
Pcb, respectively upon administration 0.25,2,8h tri- time points put to death mouse, collection tumour and testis tissue, are detected by HPLC methods
The drug concentration of its tissue.
Experimental result：Drug entities distribution results show, medicines of the Z-GP-Pcb in tumor-bearing mice testis in administration 1h
Accumulation is substantially less than Pcb (P<0.01), and Z-GP-Pcb is in administration 0.25, during Isosorbide-5-Nitrae h, the accumulation ratio of its tumour is substantially high
In Pcb (Fig. 6).
The effect experiment of the Pcb and Z-GP-Pcb anti-mouse H22 liver cancer of embodiment 6
Experimental technique：KM mouse 60 are taken, aseptic absorption is passed on the strain of H22 knurls and well-grown mouse ascites, uses physiology
Salt solution is diluted to 1*107Cells/mL, is inoculated in right side of mice armpit subcutaneous per mouse 0.2mL, sets up bearing mouse model；Inoculation
After 24h, the KM mouse that will be successfully inoculated with are randomly divided into following 6 groups, 10 per group, start within second day after modeling administration, by reagent
Thing group is administered daily, and administering mode is intraperitoneal injection 10mL/kg, and successive administration 14 days puts to death mouse after last dose 24h
And detect index of correlation.
Test index：The next day weigh the weight of animals；Eyeball takes blood after last dose, and Hematometer calculates leucocyte, blood red egg
In vain, blood platelet etc.；Knurl weight and tumour inhibiting rate：Take animal tumor tissue and claim knurl weight；
It is calculated as follows tumour inhibiting rate：
Tumour inhibiting rate=(model group knurl weight-administration group knurl weight)/model group knurl weight × 100%
Organ index：Liver, thymus gland and spleen are taken, liver, thymus gland and spleen weight index is calculated as follows：
Organ index=organ weights (mg)/body weight (g)
Experimental result：Knowable in Fig. 7, table 3 and table 4, Pcb high doses, Pcb low dosages, Z-GP-Pcb high doses, Z-
GP-Pcb low dose group tumour inhibiting rates are respectively：73.91%th, 33.93%, 69.81%, 35.39%, from test medicine tumour inhibiting rate
See, this 2 test medicines all show reasonable antitumor action, Pcb the and Z-GP-Pcb tumor killing effects of equimolar dosage
Quite, in addition in addition to Pcb high dose groups the weight of animals declines substantially, other each group body weight do not have significant changes；Pcb and Z-GP-
Two samples of Pcb affect little to index and spleen index and thymus index；Pcb high and low doses can also make animal white blood cell count(WBC), blood
Platelet content is reduced, hence it is evident that produce bone marrow inhibition；And Z-GP-Pcb high and low doses have no significant effect to haemocyte quantity；
Illustrate that Z-GP-Pcb is substantially reduced to non-target organ toxicity, improve Pcb and cause leucocyte and thrombocytopenic bad reaction.
Impacts of table 3 Pcb, the Z-GP-Pcb to H22 liver cancer tumor-bearing mices
Note：Compare with model group (reduction), * P ＜ 0.05, * * P ＜ 0.01
Impacts of the Pcb of table 4 and Z-GP-Pcb to H22 liver cancer tumor-bearing mice haemocytes
Note：Compare with model group (reduction), * P ＜ 0.05；* P ＜ 0.01；Z-GP-Pcb low dose groups are low with corresponding Pcb
Dosage group compares (rising),#P ＜ 0.05；Z-GP-Pcb high dose groups compare (rising) with corresponding Pcb high dose groups,##P ＜
0.01
Result above discloses Z-GP-Pcb of the present invention and can significantly reduce the toxicity and toxicity in vivo of normal cell,
In vivo external enwergy discharges procarbazine, of the present inventionization by FAP-alpha enzyme spcificitys hydrolysis excision dipeptide moieties (Z-GP)
Compound can significantly inhibit the growth of tumor bearing nude mice in-vivo tumour and reduce to non-target organ toxicity, be expected to develop into new drug.
It is last to should be noted that above example only to illustrate technical scheme rather than protect to the present invention
The restriction of shield scope, although being explained in detail to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (10)
1. a kind of antitumoral compounds of targeting F Α P-alpha enzymes, it is characterised in that its structure is shown in formula I：
Or shown in Formulas I compound isomer, its structure is as shown in Formula II：
2. a kind of preparation method of compound as claimed in claim 1, it is characterised in that the method comprising the steps of：
(1) by procarbazine, benzyloxycarbonyl group glycyl proline and condensation reagent, stir and react at 0 DEG C～30 DEG C, obtain mixed
Compound；
(2) after completion of the reaction, saturated salt solution is added in step (1) gained mixture, is quenched, is separated, is purified, obtained final product
Compound as claimed in claim 1.
3. the preparation method of compound as claimed in claim 2, it is characterised in that the condensation reagent in the step (1) is 1-
Hydroxybenzotriazole and N, N- diisopropylethylamine and 2- (7- aoxidizes BTA)-N, N, N', N'- tetramethylurea hexafluoro phosphorus
Acid esters, ethyl chloroformate, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, N, N '-diisopropyl carbon two is sub-
Amine, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- chlorine N, at least one in N, 2- trimethacrylate amine
Mixture.
4. as described in Claims 2 or 3 compound preparation method, it is characterised in that procarbazine, benzyl in the step (1)
The mol ratio of oxygen carbonyl glycyl proline and condensation reagent is 1:(1～3.0):(1.05～3.0).
5. the preparation method of compound as claimed in claim 2, it is characterised in that the procarbazine in the step (1) is adopted
Following method is prepared from：
A p-methylbenzoic acid is dissolved in organic reagent by (), add excessive thionyl chloride, in 60-80 DEG C of oil bath heating backflow 1-3h,
Revolving removes excessive thionyl chloride, obtains to methyl benzoyl chloride, then methyl benzoyl chloride will be redissolved in organic reagent,
Isopropylamine is slowly added to, at 30 DEG C, charging is finished and continues that reaction 2-4h is stirred at room temperature control temperature, then heats to 40
DEG C, continuing to react 20-40min, reaction is isolated and purified after terminating, and obtains N- isopropyls to methyl benzamide；
B () will be dissolved in organic reagent obtained by step (a) to methyl benzoyl chloride, be slowly added to ammonium ceric nitrate suspension, charging
Finish, be placed in 90-100 DEG C of oil bath pan and react 20-30h, reaction is isolated and purified after terminating, and obtains 4- formoxyl-N- cumenes
Formamide；
C 4- formoxyl-N- isopropyls obtained by step (b) are dissolved in organic examination by () to methyl benzamide and methylhydrazinium sulphate
Agent, adds triethylamine, reaction unit is placed in 50-70 DEG C of oil bath pan and reacts 5-7h, is spin-dried for solvent, and mixture is redissolved in
In organic reagent, sodium cyanoborohydride is slowly added in 0 DEG C of cryostat, charging is finished, and reaction system is gradually increased to
Room temperature, overnight, reaction is isolated and purified after terminating, and obtains final product procarbazine for reaction.
6. the preparation method of compound as claimed in claim 5, it is characterised in that the organic solvent in the step (a) is dry
Dry dichloromethane or the mol ratio of chloroform, the p-methylbenzoic acid and thionyl chloride is 1.0:(5.0～100), it is described right
Methyl benzoic acid is 1 with the mol ratio of isopropylamine:(5.0～10).
7. the preparation method of compound as claimed in claim 5, it is characterised in that the organic solvent in the step (b) is nitre
Acid solution or acetum, in the ammonium ceric nitrate suspension concentration of salpeter solution be 3.0-5.0mmol/L, the N- isopropyls
Base is 1 to the molar feed ratio of methyl benzamide and cerous nitrate:(3.0～5.0).
8. the preparation method of compound as claimed in claim 5, it is characterised in that the organic solvent in the step (c) is nothing
Water-ethanol or DMF, the 4- formoxyls-N- isopropylbenzamides are 1 with the mol ratio of methylhydrazinium sulphate:(3.0～
5.0)。
9. a kind of antitumoral compounds of targeting F Α P-alpha enzymes, it is characterised in that the compound is such as claim 1 institute
State the derivative or physiologically acceptable salt of compound.
10. application of the compound in antineoplastic is prepared as described in claim 1 or 9.
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| CN108864250A (en) * | 2018-05-29 | 2018-11-23 | 北京大学 | A kind of gemcitabine pro-drug and its preparation method and application of FAP α enzyme sensitivity |
| CN110156629A (en) * | 2019-05-30 | 2019-08-23 | 广州药本君安医药科技股份有限公司 | The synthetic method of procarbazine |
| CN114163478A (en) * | 2021-12-15 | 2022-03-11 | 北京师范大学 | Technetium-99 m labeled D-proline modified FAPI derivative and preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864250A (en) * | 2018-05-29 | 2018-11-23 | 北京大学 | A kind of gemcitabine pro-drug and its preparation method and application of FAP α enzyme sensitivity |
| CN110156629A (en) * | 2019-05-30 | 2019-08-23 | 广州药本君安医药科技股份有限公司 | The synthetic method of procarbazine |
| CN114163478A (en) * | 2021-12-15 | 2022-03-11 | 北京师范大学 | Technetium-99 m labeled D-proline modified FAPI derivative and preparation method and application thereof |
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