CN107400109A - The impurity A of Raltitrexed and its preparation and application - Google Patents
The impurity A of Raltitrexed and its preparation and application Download PDFInfo
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- CN107400109A CN107400109A CN201710810407.4A CN201710810407A CN107400109A CN 107400109 A CN107400109 A CN 107400109A CN 201710810407 A CN201710810407 A CN 201710810407A CN 107400109 A CN107400109 A CN 107400109A
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Abstract
The invention discloses a kind of potential Toxicity in blood system impurity A of Raltitrexed, its preparation method and its purposes as impurity reference substance.
Description
Technical field
The invention belongs to medicinal chemistry art, in particular to the preparation and its application of a kind of new impurity of Raltitrexed.
Background technology
Raltitrexed (Raltitrexed) is the water-soluble quinazoline folic acid class of new generation developed by Zeneca companies of Britain
Like thing thymidilate synthase inhibitors, suitable for advanced colorectal cancer, it is anti-by being produced to the single-minded suppression of thymidylate synthase
Function of tumor.Entitled N- [[5- [[(1,4- dihydro -2- methyl -4- oxygen -6- quinazolyls) methyl] the methylamino] -2- of its chemistry
Thienyl] carbonyl]-Pidolidone.Structural formula is as follows：
It is compared with 5 FU 5 fluorouracil, and due to special selectivity, toxic side effect substantially reduces, but it still has one
Fixed toxicity.According to relevant clinical test result show, its major toxicity adverse reaction including to intestines and stomach, hematological system and
The invertibity of liver enzyme influences.
Wherein, for the main relevant adverse reaction of hematological system, for Neuroleptic Leukocytopenia, (particularly neutrophil leucocyte subtracts
Less), anaemia and decrease of platelet (incidence is respectively 22%, 18% and 5%), can individually occur or occur simultaneously, these are anti-
It should be usually mild to moderate (WHO1/2), occur after medication in the 1st or 2 week, recover before the 3rd week.It is also possible to that severe occurs
The decrease of platelet of (WHO3/4 levels) Neuroleptic Leukocytopenia (particularly Neutrophilic granulocytopenia) and WHO4 levels, may threat to life
Or it is fatal, when especially reacting while occur with gastrointestinal toxicity.
At present when there is serious adverse reaction, clinically conventional strategy is the dosage or temporary for reducing Raltitrexed
The use of drug withdrawal product, have no the related report for how reducing Raltitrexed toxic side effect.Therefore, Raltitrexed is furtherd investigate
The reason for adverse reaction, the drug safety and validity of Raltitrexed are improved, preferably plays its clinical efficacy, reduces it not
Good reaction or side reaction, there is certain practice significance.
It has been investigated that why Raltitrexed occurs than more serious hematological system side effect, and in its finished product
Thiophene-based impurity has certain positive correlation, i.e. the content of the impurity is higher, the toxic side effect of the hematological system of Raltitrexed
Incidence is bigger.Then it is determined that the structure type of the impurity, reasonably controls its content in finished product to seem and more attaches most importance to
Will.
The content of the invention
It is an object of the invention to provide a kind of impurity for causing Raltitrexed hematological system side effect, and its preparation side
Method.
Raltitrexed is mainly made up of quinazolinone, thiophene and Pidolidone three parts, document (Quinazoline
Antifolate Thymidylate Synthase Inhibitors：Heterocyclic Benzoyl Ring
Modifications, J.Med.Chem.1991,34,1594-1605) and United States Patent (USP) (US4992550,1991) in provide
Main two methods in the synthesis of Raltitrexed, a kind of method is using 2- thiophenic acids as initiation material, synthetic route is such as
Shown in lower：
Another method is that synthetic route is as follows using 5- nitro -2- thiophenic acids as initiation material：
But through researching and analysing find, either prepared with which kind of preparation method recited above, its finally into
Can all there is following thiophene-based impurity N- (5- methylamino -2- thenoyls)-Pidolidone in product, its structure is as follows：
The impurity is caused byproduct of reaction in Raltitrexed preparation technology, with being ultimately conducted into for reaction
In product.
It is an object of the invention to provide impurity of said structure and preparation method thereof, and reasonably control its content.
A kind of preparation method and applications of Raltitrexed impurity A N- (5- methylamino -2- thenoyls)-Pidolidone.
Detailed synthetic method is as follows：
Specifically comprise the following steps：
(1) 5- nitro -2- thiophene chlorides are prepared through chlorination in 5- nitros -2- thiophenic acids；Wherein, chloro tries
Agent is selected from：Oxalyl chloride, thionyl chloride, POCl3, phosphorus trichloride, phosphorus pentachloride etc., preferably thionyl chloride；
(2) for 5- nitros -2- thiophene chlorides with Pidolidone diethylester in aprotic solvent, it is anti-that organic base does acid binding agent
N- (5- nitro -2- thenoyls)-Pidolidone diethylester should be obtained；
Wherein, aprotic solvent is selected from：Dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran equal solvent, preferably two
Chloromethanes；
Organic base is selected from：Triethylamine, diethylamine, diisopropyl ethyl amine etc., preferably triethylamine；
(3) N- (5- nitro -2- thenoyls)-Pidolidone diethylesters issue raw reduction reaction in reducing agent effect and prepared
Obtain N- (5- amino -2- thenoyls)-Pidolidone diethylester；
Wherein, reducing agent system is selected from Pd-C/H2, Fe/FeSO4, Fe/ glacial acetic acid, Pd-C/ ammonium formates, preferably Fe/ ice vinegar
Acid；
(4) N- (5- amino -2- thenoyls)-Pidolidone diethylester in polar aprotic solvent with methylating examination
Agent occurs methylation reaction and N- (5- methylamino -2- thenoyls)-Pidolidone diethylester is prepared；
Wherein, polar aprotic solvent be selected from DMF (DMF), DMA (DMA),
Acetonitrile, acetone, dimethyl sulfoxide (DMSO) etc., preferably DMF；Methylating reagent be selected from iodomethane, dimethyl suflfate,
Diazomethane etc., preferably iodomethane；
(5) N- (5- methylamino -2- thenoyls)-Pidolidone diethylesters hydrolyze under inorganic base aqueous solution effect, with
Salt acid for adjusting pH is evaporated off solvent, impurity A sterling is obtained after desalination to 3~4；
Wherein, inorganic base is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide etc., preferably sodium hydroxide.
The present invention also provides a kind of pharmaceutical composition of Raltitrexed, and the content of the Raltitrexed is excellent not less than 98%
Choosing is not less than 99%, more desirably not less than 99.8%.
Further, the content of Raltitrexed impurity A N- (5- methylamino -2- thenoyls)-Pidolidone is not higher than
0.3%, preferably no greater than 0.2%, more preferably no higher than 0.1%.
Compared with prior art, the invention has the advantages that：
1) a kind of thiophene-based impurity of Raltitrexed is disclosed, and its activity is studied, its toxicity and to making
The influence of blood system is far longer than Raltitrexed；
2) preparation method of above-mentioned impurity is disclosed, this method can obtain quality without high temperature, compressive reaction by this law
Stable, the higher impurity A reference substance of yield, foundation and clinical application security for the quality standard of Raltitrexed provide guarantor
Barrier.
3) present invention is had found by furtheing investigate, when by the control of thiophene-based impurity content 0.3% and its it is following when, can be with
The effective generation for reducing seriousness hematological system side effect.
Brief description of the drawings
The ESI-Ms spectrograms of Fig. 1 Raltitrexed impurity As；
Fig. 2 Raltitrexed impurity As1H NMR spectras；
The HPLC collection of illustrative plates of Fig. 3 Raltitrexed impurity As.
Embodiment
Below by embodiment, the invention will be further described.It should be understood that the embodiment of the present invention is only for
Illustrate the present invention, rather than limitation of the present invention, the simple modifications of the present invention are belonged under the concept thereof of the present invention
The scope of protection of present invention.
The preparation of the impurity A of embodiment 1
1. the preparation of 5- nitro -2- thiophene chlorides
10g 5- nitro -2- thiophenic acids and 29.1ml thionyl chlorides, temperature rising reflux reaction 3 are added in 50ml three-necked bottles
~4 hours, (n-hexane to terminal was reacted in TLC monitorings:Ethyl acetate=1:1).Reaction solution is transferred in 100ml single port bottles and subtracted
Pressure is concentrated into dripless, and not purified to be directly used in next step, yield is based on 100%.
The preparation of N- 2. (5- nitro -2- thenoyls)-Pidolidone diethylester
130ml dichloromethane, 19.0g Pidolidones diethylester and 51.5ml triethylamines are added in 250ml three-necked bottles,
Stirring is cooled to 0~5 DEG C, and warm in control≤5 DEG C, the dichloromethane (26ml) that 5- nitro -2- thiophene chlorides are slowly added dropwise is molten
Liquid, drop finish, are warming up to and reaction are stirred at room temperature 0~1 hour, and (n-hexane to terminal is reacted in TLC monitorings:Ethyl acetate=1:1).Instead
It should finish, reaction solution is averagely washed at twice with 150ml 2N hydrochloric acid, then is averagely washed at twice with 150ml saturated sodium bicarbonates
Wash, organic phase anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 25g.It is not purified be directly used in it is next
Step.
The preparation of N- 3. (5- amino -2- thenoyls)-Pidolidone diethylester
30ml acetonitriles, 20ml water, 18ml glacial acetic acid and 9.3g reduction Fe powder, stir-activating are added in 250ml three-necked bottles
It is 10~15 minutes, warm in control≤50 DEG C, the second of 10.0g N- (5- nitro -2- thenoyls)-L- glutamate diethyl esters is added dropwise
Nitrile (30ml) solution, drop finish, and are warming up to 70~80 DEG C and react 1~2 hour, and (n-hexane to terminal is reacted in TLC monitorings:Acetic acid second
Ester=1:1.5).10~30 DEG C are cooled to, pads one layer of suction filtered through kieselguhr, filter cake is eluted with 20ml acetonitriles, filtered to dripless.Filter
Liquid is concentrated under reduced pressure in 60~70 DEG C, and the average 50ml toluene band that adds at twice removes glacial acetic acid, is concentrated into dripless.Residue adds
30ml ethyl acetate, it is sufficiently stirred 10~15 minutes, filters, 30ml water is added in filtrate, and adjusted with 20% sodium carbonate liquor
PH to 8~9, liquid separation is stirred, separates organic phase, aqueous phase is extracted twice with 30ml*2 ethyl acetate again, merges organic phase, anhydrous
Sodium sulphate is dried.Filter, be concentrated under reduced pressure to obtain brown oil 8.5g.
Above-mentioned grease is dissolved with ethyl acetate, post is filled with 11.7g silica gel sand, 97.8g silica gel, with n-hexane/acetic acid
Ethyl ester=3/1 elutes, and TLC is shown as collecting eluent during unique spot, obtains pale yellow oil, i.e. N- (5- amino -2- thiophene
Formyl)-Pidolidone diethylester, weight 6.5g.
The preparation of N- 4. (5- methylamino -2- thenoyls)-Pidolidone diethylester
5g N- (5- amino -2- thenoyls)-Pidolidone diethylester, 57.7ml N, N- are added in 100ml three-necked bottles
Dimethylformamide, 1.4ml 2,6- lutidines and 2.68g iodomethane, it is warming up to 45~48 DEG C and stirs 6 hours, then heats up
Reacted 6 hours to 50~52 DEG C.Stop reaction, be cooled to 10~30 DEG C.38.6ml water is added, is averaged with 120ml ethyl acetate
Extract in three times, merge organic phase, anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 6.23g.
Above-mentioned grease is dissolved with ethyl acetate, post is filled with 8.15g silica gel sand, 67.9g silica gel, with n-hexane/acetic acid
Ethyl ester=3/1 elutes, and TLC is shown as collecting eluent during unique spot, obtains pale yellow oil, i.e. N- (5- methylamino -2- thiophenes
Fen formyl)-Pidolidone diethylester, weight 3.5g.
The preparation of N- 5. (5- methylamino -2- thenoyls)-Pidolidone
5.2g sodium hydroxides and 85ml water are added in 100ml reaction bulbs, is cooled to 10-15 DEG C in advance, add N- (5- methylaminos-
2- thenoyls)-Pidolidone diethylester, the stirring reaction 3-5 hours at 10-15 DEG C, TLC monitoring reactions are complete, add
30ml ethyl acetate extracts 3 times, and aqueous phase is cooled to 10-15 DEG C again, adjusts pH to 3-4 with concentrated hydrochloric acid, reaction is stirred overnight.It is next
Day, at 60 DEG C, decompression is spin-dried for solvent, and residue adds the mashing of 100ml methanol, filtered, and at 60 DEG C of filtrate, decompression is spin-dried for solvent,
Residue adds 60ml acetone mashing 30min, and suction filtration dries to obtain compound 2.1g, yield 81%, and HPLC (is referred to attached for 96.7%
Fig. 3).1HNMR, (d6-DMSO, 500M) δ：1.8728-1.9330 (2H, m)；2.1833-2.2253 (2H, m)； 2.7406-
207497 (3H, d)；4.1824 (1H, s)；5.8007-5.8088 (1H, d)；6.8378-6.8476 (1H, d)；7.3713-
7.3794 (1H, d)；7.5771 (1H, s)；MS:found：285.1, [M-H]+(referring to accompanying drawing 1,2).
The preparation of the impurity A of embodiment 2
1. the preparation of 5- nitro -2- thiophene chlorides
10g 5- nitro -2- thiophenic acids and 29.1ml POCl3s, temperature rising reflux reaction 3 are added in 50ml three-necked bottles
~4 hours, (n-hexane to terminal was reacted in TLC monitorings:Ethyl acetate=1:1).Reaction solution is transferred in 100ml single port bottles and subtracted
Pressure is concentrated into dripless, and not purified to be directly used in next step, yield is based on 100%.
The preparation of N- 2. (5- nitro -2- thenoyls)-Pidolidone diethylester
130ml chloroforms, 19.0g Pidolidones diethylester and 51.5ml diethylamine are added in 250ml three-necked bottles,
Stirring is cooled to 0~5 DEG C, and warm in control≤5 DEG C, the dichloromethane (26ml) that 5- nitro -2- thiophene chlorides are slowly added dropwise is molten
Liquid, drop finish, are warming up to and reaction are stirred at room temperature 0~1 hour, and (n-hexane to terminal is reacted in TLC monitorings:Ethyl acetate=1:1).Instead
It should finish, reaction solution is averagely washed at twice with 150ml 2N hydrochloric acid, then is averagely washed at twice with 150ml saturated sodium bicarbonates
Wash, organic phase anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 24.6g.It is not purified be directly used in it is next
Step.
The preparation of N- 3. (5- amino -2- thenoyls)-Pidolidone diethylester
Add methanol 80ml in 250ml three-necked bottles, 5%Pd/C, after nitrogen displacement 3 times, hydrogen is replaced 3 times, in hydrogen
Stir-activating 10~15 minutes under (1atm) atmosphere, it is warm in control≤30 DEG C, 10.0g N- (5- nitro -2- thiophene first is added dropwise
Acyl)-Pidolidone diethylester methanol (30ml) solution, drop finish, react at room temperature 1~2 hour, TLC monitoring reaction to terminal
(n-hexane:Ethyl acetate=1:1.5).10~30 DEG C are cooled to, pads one layer of suction filtered through kieselguhr, filter cake is eluted with 20ml methanol,
Filter to dripless.Filtrate is concentrated under reduced pressure in 40~50 DEG C, is concentrated into dripless, obtains brown oil 8.3g.
Above-mentioned grease is dissolved with ethyl acetate, is filled post with 15g silica gel sand, 83g silica gel, is used n-hexane/ethyl acetate
=3/1 elution, TLC are shown as collecting eluent during unique spot, obtain pale yellow oil, i.e. N- (5- amino -2- thiophene first
Acyl)-Pidolidone diethylester, weight 5.7g.
The preparation of N- 4. (5- methylamino -2- thenoyls)-Pidolidone diethylester
5g N- (5- amino -2- thenoyls)-Pidolidone diethylester, 57.7ml N, N- are added in 100ml three-necked bottles
Dimethyl acetamide, 1.4ml 2,6- lutidines and 1.5g dimethyl suflfates, it is warming up to 45~48 DEG C and stirs 6 hours, then
50~52 DEG C are warming up to react 6 hours.Stop reaction, be cooled to 10~30 DEG C.38.6ml water is added, with 120ml ethyl acetate
It is average to extract in three times, merge organic phase, anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 6.31g.
Above-mentioned grease is dissolved with ethyl acetate, post is filled with 10g silica gel sand, 63.1g silica gel, with n-hexane/acetic acid second
Ester=3/1 elutes, and TLC is shown as collecting eluent during unique spot, obtains pale yellow oil, i.e. N- (5- methylamino -2- thiophenes
Fen formyl)-Pidolidone diethylester, weight 2.6g.
The preparation of N- 5. (5- methylamino -2- thenoyls)-Pidolidone
4.6g potassium hydroxide and 85ml water are added in 100ml reaction bulbs, is cooled to 10-15 DEG C in advance, add N- (5- methylaminos-
2- thenoyls)-Pidolidone diethylester, the stirring reaction 3-5 hours at 10-15 DEG C, TLC monitoring reactions are complete, add
30ml ethyl acetate extracts 3 times, and aqueous phase is cooled to 10-15 DEG C again, adjusts pH to 3-4 with concentrated hydrochloric acid, reaction is stirred overnight.It is next
Day, at 60 DEG C, decompression is spin-dried for solvent, and residue adds the mashing of 100ml methanol, filtered, and at 60 DEG C of filtrate, decompression is spin-dried for solvent,
Residue adds 60ml acetone mashing 30min, and suction filtration dries to obtain compound 1.7g, yield 75.3%.
The preparation of the impurity A of embodiment 3
1. the preparation of 5- nitro -2- thiophene chlorides
10g 5- nitro -2- thiophenic acids and 29.1ml phosphorus pentachlorides, temperature rising reflux reaction 3 are added in 50ml three-necked bottles
~4 hours, (n-hexane to terminal was reacted in TLC monitorings:Ethyl acetate=1:1).Reaction solution is transferred in 100ml single port bottles and subtracted
Pressure is concentrated into dripless, and not purified to be directly used in next step, yield is based on 100%.
The preparation of N- 2. (5- nitro -2- thenoyls)-Pidolidone diethylester
130ml tetrahydrofurans, 19.0g Pidolidones diethylester and 51.5ml diisopropyls are added in 250ml three-necked bottles
Ethylamine, stirring are cooled to 0~5 DEG C, warm in control≤5 DEG C, and the dichloromethane of 5- nitro -2- thiophene chlorides is slowly added dropwise
(26ml) solution, drop finish, are warming up to and reaction are stirred at room temperature 0~1 hour, and (n-hexane to terminal is reacted in TLC monitorings:Ethyl acetate
=1:1).Reaction is finished, and reaction solution is averagely washed at twice with 150ml 2N hydrochloric acid, then is averaged with 150ml saturated sodium bicarbonates
Wash at twice, organic phase anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 24.8g.It is not purified direct
For in next step.
The preparation of N- 3. (5- amino -2- thenoyls)-Pidolidone diethylester
30ml acetonitriles, 20ml water, 18ml glacial acetic acid and 9.3g reduction Fe powder, stir-activating are added in 250ml three-necked bottles
It is 10~15 minutes, warm in control≤50 DEG C, the second of 10.0g N- (5- nitro -2- thenoyls)-L- glutamate diethyl esters is added dropwise
Nitrile (30ml) solution, drop finish, and are warming up to 70~80 DEG C and react 1~2 hour, and (n-hexane to terminal is reacted in TLC monitorings:Acetic acid second
Ester=1:1.5).10~30 DEG C are cooled to, pads one layer of suction filtered through kieselguhr, filter cake is eluted with 20ml acetonitriles, filtered to dripless.Filter
Liquid is concentrated under reduced pressure in 60~70 DEG C, and the average 50ml toluene band that adds at twice removes glacial acetic acid, is concentrated into dripless.Residue adds
30ml ethyl acetate, it is sufficiently stirred 10~15 minutes, filters, 30ml water is added in filtrate, and adjusted with 20% sodium carbonate liquor
PH to 8~9, liquid separation is stirred, separates organic phase, aqueous phase is extracted twice with 30ml*2 ethyl acetate again, merges organic phase, anhydrous
Sodium sulphate is dried.Filter, be concentrated under reduced pressure to obtain brown oil 7.9g.
Above-mentioned grease is dissolved with ethyl acetate, post is filled with 14.2g silica gel sand, 79g silica gel, with n-hexane/acetic acid second
Ester=3/1 elutes, and TLC is shown as collecting eluent during unique spot, obtains pale yellow oil, i.e. N- (5- amino -2- thiophene
Formyl)-Pidolidone diethylester, weight 6.3g.
The preparation of N- 4. (5- methylamino -2- thenoyls)-Pidolidone diethylester
Addition 5g N- (5- amino -2- thenoyls)-Pidolidone diethylester in 100ml three-necked bottles, 57.7ml acetonitriles,
1.4ml 2,6- lutidines and 2.68g iodomethane, it is warming up to 45~48 DEG C and stirs 6 hours, then is warming up to 50~52 DEG C instead
Answer 6 hours.Stop reaction, be cooled to 10~30 DEG C.38.6ml water is added, is averagely extracted in three times with 120ml ethyl acetate,
Merge organic phase, anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 6.1g.
Above-mentioned grease is dissolved with ethyl acetate, is filled post with 12g silica gel sand, 61g silica gel, is used n-hexane/ethyl acetate
=3/1 elution, TLC are shown as collecting eluent during unique spot, obtain pale yellow oil, i.e. N- (5- methylamino -2- thiophene
Formyl)-Pidolidone diethylester, weight 3.4g.
The preparation of N- 5. (5- methylamino -2- thenoyls)-Pidolidone
5.2g sodium hydroxides and 85ml water are added in 100ml reaction bulbs, is cooled to 10-15 DEG C in advance, add N- (5- methylaminos-
2- thenoyls)-Pidolidone diethylester, the stirring reaction 3-5 hours at 10-15 DEG C, TLC monitoring reactions are complete, add
30ml ethyl acetate extracts 3 times, and aqueous phase is cooled to 10-15 DEG C again, adjusts pH to 3-4 with concentrated hydrochloric acid, reaction is stirred overnight.It is next
Day, at 60 DEG C, decompression is spin-dried for solvent, and residue adds the mashing of 100ml methanol, filtered, and at 60 DEG C of filtrate, decompression is spin-dried for solvent,
Residue adds 60ml acetone mashing 30min, and suction filtration dries to obtain compound 1.8g, yield 76.5%.
The preparation of the impurity A of embodiment 4
1. the preparation of 5- nitro -2- thiophene chlorides
Addition 10g 5- nitro -2- thiophenic acids and 29.1ml oxalyl chlorides in 50ml three-necked bottles, temperature rising reflux reaction 3~
4 hours, (n-hexane to terminal was reacted in TLC monitorings:Ethyl acetate=1:1).Reaction solution is transferred in 100ml single port bottles and depressurized
Dripless is concentrated into, not purified to be directly used in next step, yield is based on 100%.
The preparation of N- 2. (5- nitro -2- thenoyls)-Pidolidone diethylester
130ml tetrahydrofurans, 19.0g Pidolidones diethylester and 51.5ml diethylamine are added in 250ml three-necked bottles,
Stirring is cooled to 0~5 DEG C, and warm in control≤5 DEG C, the dichloromethane (26ml) that 5- nitro -2- thiophene chlorides are slowly added dropwise is molten
Liquid, drop finish, are warming up to and reaction are stirred at room temperature 0~1 hour, and (n-hexane to terminal is reacted in TLC monitorings:Ethyl acetate=1:1).Instead
It should finish, reaction solution is averagely washed at twice with 150ml 2N hydrochloric acid, then is averagely washed at twice with 150ml saturated sodium bicarbonates
Wash, organic phase anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 21.6g.It is not purified be directly used in it is next
Step.
The preparation of N- 3. (5- amino -2- thenoyls)-Pidolidone diethylester
30ml acetonitriles, 20ml water, 18ml ferrous sulfate and 9.3g reduction Fe powder, stirring is added in 250ml three-necked bottles to live
Change 10~15 minutes, it is warm in control≤50 DEG C, 10.0g N- (5- nitro -2- thenoyls)-L- glutamate diethyl esters are added dropwise
Acetonitrile (30ml) solution, drop finish, and are warming up to 70~80 DEG C and react 1~2 hour, and (n-hexane to terminal is reacted in TLC monitorings:Acetic acid
Ethyl ester=1:1.5).10~30 DEG C are cooled to, pads one layer of suction filtered through kieselguhr, filter cake is eluted with 20ml acetonitriles, filtered to dripless.
Filtrate is concentrated under reduced pressure in 60~70 DEG C, and the average 50ml toluene band that adds at twice removes glacial acetic acid, is concentrated into dripless.Residue adds
Enter 30ml ethyl acetate, be sufficiently stirred 10~15 minutes, filter, 30ml water is added in filtrate, and adjusted with 20% sodium carbonate liquor
PH to 8~9 is saved, liquid separation is stirred, separates organic phase, aqueous phase is extracted twice with 30ml*2 ethyl acetate again, merges organic phase, nothing
Aqueous sodium persulfate is dried.Filter, be concentrated under reduced pressure to obtain brown oil 8.5g.
Above-mentioned grease is dissolved with ethyl acetate, is filled post with 15g silica gel sand, 85g silica gel, is used n-hexane/ethyl acetate
=3/1 elution, TLC are shown as collecting eluent during unique spot, obtain pale yellow oil, i.e. N- (5- amino -2- thiophene first
Acyl)-Pidolidone diethylester, weight 7.3g.
The preparation of N- 4. (5- methylamino -2- thenoyls)-Pidolidone diethylester
Addition 5g N- (5- amino -2- thenoyls)-Pidolidone diethylester in 100ml three-necked bottles, 57.7ml acetonitriles,
1.4ml 2,6- lutidines and 2.68g iodomethane, it is warming up to 45~48 DEG C and stirs 6 hours, then is warming up to 50~52 DEG C instead
Answer 6 hours.Stop reaction, be cooled to 10~30 DEG C.38.6ml water is added, is averagely extracted in three times with 120ml ethyl acetate,
Merge organic phase, anhydrous sodium sulfate drying.Filter, be concentrated under reduced pressure to obtain brown oil 5.8g.
Above-mentioned grease is dissolved with ethyl acetate, is filled post with 12g silica gel sand, 58g silica gel, is used n-hexane/ethyl acetate
=3/1 elution, TLC are shown as collecting eluent during unique spot, obtain pale yellow oil, i.e. N- (5- methylamino -2- thiophene
Formyl)-Pidolidone diethylester, weight 2.8g.
The preparation of N- 5. (5- methylamino -2- thenoyls)-Pidolidone
5.2g sodium hydroxides and 85ml water are added in 100ml reaction bulbs, is cooled to 10-15 DEG C in advance, add N- (5- methylaminos-
2- thenoyls)-Pidolidone diethylester, the stirring reaction 3-5 hours at 10-15 DEG C, TLC monitoring reactions are complete, add
30ml ethyl acetate extracts 3 times, and aqueous phase is cooled to 10-15 DEG C again, adjusts pH to 3-4 with concentrated hydrochloric acid, reaction is stirred overnight.It is next
Day, at 60 DEG C, decompression is spin-dried for solvent, and residue adds the mashing of 100ml methanol, filtered, and at 60 DEG C of filtrate, decompression is spin-dried for solvent,
Residue adds 60ml acetone mashing 30min, and suction filtration dries to obtain compound 2.2g, yield 83%.
The toxicity research of the impurity A of embodiment 5
Experimental animal：SD rats 100, body weight 200g ± 20g；Kun Ming mice 100, body weight 20g ± 2g.Animal
Tail vein injection gives Raltitrexed impurity A, and dosage is：Rat is that 1.0ml/100g (1mg/ml, uses sodium chloride solution
It is diluted to prescribed concentration), mouse is 0.1ml/10g (1mg/ml, prescribed concentration being diluted to sodium chloride solution), daily administration one
It is secondary.After administration, Continuous Observation 2 weeks.
Observation index：
(1) after injecting medicine, death time of animal and its The dead quantity are observed, and calculate LD50Value；
(2) poisoning symptom and duration after being administered；
(3) dead animal stands row dissection and checked；At the end of observation, dissection inspection is carried out to surviving animals.
The LD of the Raltitrexed of table 1 and its impurity A50Value
The LD of Raltitrexed is can be seen that from upper table data50Value in SD rats and mouse for 83 times of impurity A and
55 times, illustrate that the toxicity of impurity A is much larger than the toxicity of Raltitrexed, thus speculate, its side effect has certain phase with its impurity
Guan Xing.Influence of the Raltitrexed impurity A of embodiment 6 to murine interleukin and granulocyte
Female KM kind small white mouse 60,20 ± 2g of body weight, after adapting to raising, solvent control group, thunder are randomly divided into for song
Plug group, Raltitrexed impurity A group, totally 3 groups.
Test medicine：Purity be 99.8% Raltitrexed (commercially available Raltitrexed bulk drug by Nanjing it is honest become a fine day it is limited
Responsible company's production provides), yellow crystalline powder, Raltitrexed impurity A prepared by embodiment 1, off-white powder.
Medication：Mouse tail vein injection is administered, administered volume 0.1ml/10g, successive administration 4 weeks, before administration, by
Reagent thing dissolves through 0.9% sodium chloride solution, and is diluted to required concentration according to dose requirements.
Dose design：It is (3mg/m according to people's clinical dosage of Raltitrexed2/ day), it is daily equivalent to 70kg people
0.075mg/kg, the dosage of mouse is set as 0.029mg/g using this.
The high dose of Raltitrexed impurity A is 19mg/kg, middle dosage 9mg/kg, low dosage 3mg/kg, control group
Give isometric physiological saline.
Observation index
Blood picture detects：Tail vein takes blood 20ul, to produce the detection of blood picture detector daily.Each group mouse before administration, administration phase
Between weekly, administration terminate one week respectively detect once.It the results are shown in Table 2 and table 3.
Influence (unit 10 of the Raltitrexed impurity A of table 2 to mouse peripheral blood leucocyte9/L)
* represent compared with control group, p ＜ 0.05
From table 2, compared with control group, Raltitrexed leucocyte content has declined, but Raltitrexed impurity A is each
The leukocyte count of dosage group animal declines all larger, the presentation significant difference compared with control group.Illustrate that Raltitrexed impurity A can
The leukocyte count of mouse peripheral blood is caused to reduce, and influence of the Raltitrexed to peripheral leukocytes in itself is smaller, therefore rationally control miscellaneous
Matter content can effectively reduce the generation of its serious blood system side effect.
Influence of the Raltitrexed impurity A of table 3 to mouse granulocyte
From table 3, compared with control group, the mouse bone marrow cells of Raltitrexed group are in slightly suppressed state, but grain is thin
The total ratio change of born of the same parents' system is little, and the proportion of composing of grain system variant differential period does not also occur notable difference；And thunder
Marrow for bent plug impurity A group mouse is suppressed state in serious, and the proportion of composing of grain system variant differential period is also
There is notable difference.Illustrate that generation that Raltitrexed impurity A can be to mouse granulocyte and differentiation generation have a strong impact on.
To sum up, it can be seen that influence and toxicity of the Raltitrexed impurity A for mouse blood system are significantly greater than thunder
For Qu Sai, therefore its content in final finished is rationally controlled, can effectively reduce the serious side effects related to hematological system
Occur, so as to lift the security that Raltitrexed is clinically applied.The Raltitrexed composition of embodiment 7 is to mouse granulocyte
Influence
Female KM kind small white mouse 80,20 ± 2 grams of body weight, after adapting to raising, solvent control group, thunder is randomly divided into for song
Plug group, Raltitrexed and Raltitrexed impurity A group, totally 8 groups.
Test medicine：Purity be 99.8% Raltitrexed, yellow crystalline powder；Raltitrexed and Raltitrexed impurity A
Composition, wherein impurity A accounts for the weight ratio respectively 0.1% of composition, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%
(calling in the following text " composition ").
Before administration, test medicine dissolves through water for injection, and according to dosage requires to be diluted to required concentration.
Medication：Mouse tail vein injection is administered, and administered volume is 0.1ml/10g body weight, successive administration 4 weeks.
Dose design：It is (3mg/m according to people's clinical dosage of Raltitrexed2/ day), it is daily equivalent to 70kg people
0.075mg/kg, the dosage of mouse was set as 0.029mg/ days using this, control group gives isometric physiological saline.
Observation index
Blood picture detects：Tail vein takes the μ l of blood 20, to produce the detection of blood picture detector daily.Each group mouse before administration, administration phase
Between weekly, administration terminate one week respectively detect once.It the results are shown in Table 4.
Influence of the Raltitrexed of table 4 and combinations thereof to mouse bone marrow cells smear cells differential counting result
From table 4, the marrow of Raltitrexed group mouse becomes in slightly suppressed state, the total ratio of granulocytic systen
Change less, and the proportion of composing of grain system variant differential period does not also occur notable difference；And the performance of composition group is different, when
During the content of Raltitrexed impurity A in composition≤0.20, the marrow of each group mouse is in slight holddown, granulocytic systen
Total ratio change is little, and the proportion of composing of grain system variant differential period does not also occur notable difference and Raltitrexed class
Seemingly；It is small with the increase of Impurity A content when the content of Raltitrexed impurity A in composition reaches 0.4%, 0.5% and 0.6%
The marrow of mouse aggravates all the more in the state of being suppressed, and the difference that also occurs of the proportion of composing of grain system variant differential period by
It is cumulative big, illustrate generation that Raltitrexed composition can be to granulocyte and breaks up generation to have a strong impact on.Therefore, in order to ensure to use
The security of medicine, the content of Raltitrexed impurity A should not be greater than 0.30%.
Claims (10)
1. the impurity A of Raltitrexed, it is characterised in that there is following structure：
。
2. a kind of preparation method of Raltitrexed impurity A, it is characterised in that reaction scheme is as follows：
Specifically include following steps：
1) 5- nitro -2- thiophene chlorides are prepared through chlorination in 5- nitros -2- thiophenic acids；
2) with Pidolidone diethylester in aprotic solvent, organic base does acid binding agent and reacted 5- nitros -2- thiophene chlorides
To N- (5- nitro -2- thenoyls)-Pidolidone diethylester；
3) N- (5- nitro -2- thenoyls)-Pidolidone diethylesters issue raw reduction reaction in reducing agent effect and are prepared
N- (5- amino -2- thenoyls)-Pidolidone diethylester；
4) N- (5- amino -2- thenoyls)-Pidolidone diethylester is sent out in polar aprotic solvent with methylating reagent
N- (5- methylamino -2- thenoyls)-Pidolidone diethylester is prepared in raw methylation reaction；
5) N- (5- methylamino -2- thenoyls)-Pidolidone diethylesters are hydrolyzed and are acidified under inorganic base aqueous solution effect, are obtained
To impurity A.
3. the preparation method of Raltitrexed impurity A according to claim 2, it is characterised in that the chlorine described in step 1)
It is selected from for reagent：One kind in oxalyl chloride, thionyl chloride, POCl3, phosphorus trichloride, phosphorus pentachloride.
4. the preparation method of Raltitrexed impurity A according to claim 2, it is characterised in that tiing up described in step 2)
Sour agent is selected from：Triethylamine, diethylamine, one kind in diisopropyl ethyl amine, aprotic solvent are selected from dichloromethane, three chloromethanes
Alkane, carbon tetrachloride, one kind in tetrahydrofuran.
5. the preparation method of Raltitrexed impurity A according to claim 2, it is characterised in that reduced described in step 3)
Agent system is selected from Pd-C/H2, Fe/FeSO4, Fe/ glacial acetic acid, one kind in Pd-C/ ammonium formates.
6. the preparation method of Raltitrexed impurity A according to claim 2, it is characterised in that polarity described in step 4)
Non-protonic solvent in N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, acetonitrile, acetone, dimethyl sulfoxide (DMSO) one
Kind；The one kind of methylating reagent in iodomethane, dimethyl suflfate, diazomethane.
7. the preparation method of Raltitrexed impurity A according to claim 2, it is characterised in that inorganic described in step 5)
The one kind of alkali in lithium hydroxide, sodium hydroxide, potassium hydroxide.
8. the preparation method of the Raltitrexed impurity A according to claim 2-7, it is characterised in that in the step 1)
Chlorinating agent is thionyl chloride；Acid binding agent described in step 2) is triethylamine, aprotic solvent is dichloromethane；In step 3)
The reducing agent is Fe/ glacial acetic acid；Polar aprotic solvent described in step 4) is DMF, and methylate examination
Agent is iodomethane；Inorganic base described in step 5) is sodium hydroxide.
9. the pharmaceutical composition of the Raltitrexed impurity A according to claim 1-8, it is characterised in that wherein Raltitrexed
Content is not less than 99%, more desirably not less than 99.8%, and the content of Raltitrexed impurity A is not higher than 0.2%, is preferably no greater than
0.1%.
10. purposes of the Raltitrexed impurity A in Raltitrexed Related substances separation as impurity reference substance.
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|---|---|---|---|---|
| CN114989154A (en) * | 2022-06-16 | 2022-09-02 | 杭州国瑞生物科技有限公司 | Raltitrexed dimer impurity, synthesis method and application thereof |
| CN115772165A (en) * | 2022-12-03 | 2023-03-10 | 山东百诺医药股份有限公司 | Key impurity of raltitrexed and preparation method thereof |
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| CN102898415A (en) * | 2012-10-31 | 2013-01-30 | 济南久创化学有限责任公司 | Method for preparing raltitrexed intermediate, i.e., N-(5-methylamino-2-thiophene formyl)-L-glutamate diethyl ester |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114989154A (en) * | 2022-06-16 | 2022-09-02 | 杭州国瑞生物科技有限公司 | Raltitrexed dimer impurity, synthesis method and application thereof |
| CN115772165A (en) * | 2022-12-03 | 2023-03-10 | 山东百诺医药股份有限公司 | Key impurity of raltitrexed and preparation method thereof |
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