A kind of industrialized preparing process of antibody coupling medicine connexon
Technical field
The present invention relates to a kind of industrialized preparing process preparing antibody coupling medicine Adcetris connexon.This
Bright belong to organic synthesis field.
Background technology
, as a kind of important anti-cancer therapies, the cytotoxic reagent that it is used is due to tumor for chemotherapy
Cell lacks specificity, can lead to serious side effect.And antibody coupling medicine (Antibody-Drug
Conjugate, ADC), as one kind of targeted anticancer medicine, can be by monoclonal antibody and high efficiency cell toxin
Ideally combine, that is, take full advantage of the targeting of monoclonal antibody and the advantage that selectivity is strong;
The advantage that again make use of the High Fragmentation to tumor cell for the cytotoxin well;Simultaneously because cytotoxin with
After antibody coupling, medicine that it the is formed toxicity very little to body, also solve single cell poison well
The element defect big to physical toxicity.
General ADC includes target head antibody, introns, chemical linker (Chemical Linker) and medicine
As cytotoxic compound four part.The structure that it is critical only that connexon of ADC drug development, when medicine exists
It is ensured that the antibody and chemicalses integrity under environment in vivo before entering target cell, and proximal or into
After target cell, chemicalses can accurately discharge again.Realize this process and depend on antibody and toxic chemical
" stability " after being connected by connexon.In addition the chemicalses quantity that in ADC medicine, antibody is loaded
Also depend on connexon.The failure of early stage ADC medicament research and development, is passed through with drug molecule mainly due to antibody
After connexon combines, the medicine that formed stability in vivo is poor, and toxic chemical is not reach tumor thin
Just disconnect with the antibody being connected before born of the same parents, cause the serious toxicity to body.These factors have impact on
ADC Drug safety and effectiveness.
The ADC medicine of the listing of U.S.'s food and Drug Administration (FDA) approval in the market has three
Kind, respectively Mylotarg, Adcetris and Kadcyla.Adcetris be by Seattle Genetice company and
Military field pharmacy joint development, for treating Hodgkin lymphoma and systemic primary cutaneous type.
This medicine is currently the only commercially successful ADC medicine, and it is beautiful that listing the first annual sales amount reaches 1.36 hundred million
Unit.It comprises three parts, most important of which is that one can be by antibody cAC10 and Microtubule disruption agent list
The connexon (VIII) that methyl (MMAE) connects, its structure is as follows, and this connexon passes through disulfide bond and resists
Body combines, and the medicine being connected by this mode is more stable in human body.
Its synthetic route in document Bioconjugate Chem, disclosed in 2002,13,855-869.Compound
Through condensation, deprotection, two step substitution reactions obtain connexon VIII to I.The following institute of route of report in document
Show:
The method literature procedures amount is less, only milligram rank.The yield of final step reaction only 15%, and
And post processing bothers, compound VIII needed column purification.Obviously, the method is not suitable for industry's enlarging production.
Patent US20050238649 reports and obtains compound for initiation material through four-step reaction with compound I
VIII.Although the method post-processing operation is simple, the purity of compound VIII only 94% is it is impossible to reach
The quality standard of pharmaceutical industry API.
The preparation of connexon VIII, patent US20050238649 and document Bioconjugate
Chem, 2002,13,855-869 all do not mention the physical property of intermediate, but in the middle of finding in experimentation
The easy moisture absorption of body compound IV, this undoubtedly increased the difficulty of post-reaction treatment.Based on the fact above, this
Bright hygroscopicity is improved by the method to protection on compound IV hydroxyl, simplify post-processing operation, obtain relatively
Good result.
Compound VIII, as the connexon of currently the only commercially successful ADC medicine, does not but have
Document or patent have a large amount of reports synthesizing to this compound.Therefore design and implement one and can be suitably used for industry
Metaplasia is produced, and the synthetic method that easy and simple to handle and purity can reach the connexon VIII of pharmaceutical industry standard has relatively
High economic worth and social benefit.
Content of the invention
It is an object of the invention to provide a kind of industrialized preparing process of ADC connexon, to solve above-mentioned background
The problem that technology is mentioned.
To achieve these goals, the present invention employs the following technical solutions to realize:
A kind of industrialized preparing process of ADC connexon, process route is as follows:
Wherein, R1Can be silylation protection group, Alkoxyalkyl protecting groups, alkyl protecting groups.Preferably
R1For trimethyl silicon substrate (TMS), t-Butyldimethylsilyl (TBS), alkoxy methyl protection group (MOM),
2- (trimethyl silicon substrate) ethoxyl methyl (SEM), more preferably t-Butyldimethylsilyl.
More specifically, the preparation method of the present invention comprises following process:
Step 5:Under inert gas shielding, compound I is in the presence of carboxylic acid activating reagents and alkali and chemical combination
Thing IX reaction obtains compounds X.
Wherein R1As defined above.
Described condensation reaction method and condition can be the conventional method of the such reaction in this area and condition.Relatively
Good, above-mentioned preparation method comprises the steps of:Under nitrogen protection, compound I and compound IX is added to
In organic solvent, under room temperature, stirring half an hour all dissolves to raw material, adds carboxylic acid activating reagents and alkali,
Deca process temperature controls at -5~15 DEG C.Completion of dropping, is slowly increased to room temperature and continues condensation reaction up to liquid
In phase tracing display reaction system, raw material I disappears, stopped reaction.Through normal pressure precipitation after reaction stopping, washing,
Vacuum drying obtains compounds X.
Described carboxylic acid activating reagents be selected from ethyl chloroformate, isobutyl chlorocarbonate, carbonyl dimidazoles, 4- (4,
6- dimethoxy-triazine -2- base) -4- methyl morpholine hydrochloride (DMTMM), 2- chloro- 4,6- dimethoxy -1,
3,5- triazines (CDMT), preferably ethyl chloroformate.Described carboxylic acid activating reagents and compound I rub
Your rate of charge is 1~4:1.
Described alkali is potassium acetate, pyridine, triethylamine, the organic base such as DIPEA, or potassium phosphate,
Potassium carbonate, sodium hydroxide, cesium fluoride, the inorganic base such as cesium carbonate, preferably triethylamine.Described alkali and change
The molar feed ratio of compound I is 1~6:1.
Described organic solvent is oxolane, dimethyl sulfoxide, DMF, ethyl acetate,
One of dichloromethane, or the combination in any of above-mentioned solvent, preferably ethyl acetate.Described is organic molten
The volume mass of agent and compound I is than for 5~20mL/g.
Described compound IX is commercially available.The molar feed ratio of described compound IX and compound I be 1~
4:1.
Described compound I list of references US20050238649 is obtained.
Step 6:In the basic conditions, polar non-solute is by deprotection method for compounds X
Compound XI.
Wherein R1As defined above.
Described deprotection method and condition can be the conventional method of the such reaction in this area and condition.Preferably
, above-mentioned preparation method comprises the steps of:Under room temperature, compounds X is added in polar non-solute,
Stirring half an hour all dissolves to raw material, adds alkali, carries out deprotection, until the reaction of liquid phase tracing display
In system, raw material X disappears, stopped reaction.After reaction stops, precipitation, recrystallization, washing, it is vacuum dried
To compounds X I.Described alkali is potassium acetate, pyridine, diethylamine, the organic base such as diisopropyl ethyl amine,
It is preferably diethylamine.The molar feed ratio of described alkali and compounds X is 2~5:1.
Described polar non-solute is acetonitrile, acetone, DMF or dimethyl sulfoxide
(DMSO), preferably DMF.Described polar non-solute and the body of compounds X
Long-pending mass ratio is 5~20mL/g.
Step 7:Compounds X I and compound V passes through substitution reaction, then sloughs R1Protection obtains intermediate
VI.
Wherein R1As defined above.
Described replacement and deprotection method and condition can be conventional method and the bar of the such reaction in this area
Part, preferably, above-mentioned preparation method comprises the steps of:Under room temperature, compounds X I and compound V is added to pole
Property aprotic solvent in, under greenhouse stirring half an hour to raw material all dissolve, then heat at 45~50 DEG C
Reaction 4~6 hours, obtains substituent reactant liquor, and it is de- that substituent reactant liquor is cooled to -5~0 DEG C of addition
Protection reagent, completion of dropping, it is warmed to room temperature reaction until raw material disappears in liquid phase tracing display reaction system,
Stopped reaction.After reaction stops, reactant liquor is cooled to -5~0 DEG C of tune pH value 7~8, filters, collect filter
Cake, making beating, vacuum drying obtains compound VI.Described polar non-solute is acetonitrile, acetone, N, N-
Dimethylformamide or dimethyl sulfoxide (DMSO), preferably DMF.Itself and chemical combination
The volume mass of thing XI is than for 5~20mL/g.
Described deprotecting regent can be tetrabutyl ammonium fluoride (TBAF), Methanaminium, N,N,N-trimethyl-, fluoride, cesium fluoride,
Fluohydric acid. pyridine, the fluorination reagent such as Fluohydric acid. triethylamine or p-methyl benzenesulfonic acid, trifluoroacetic acid etc. has
Machine acid, can also be hydrochloric acid, sulphuric acid, the mineral acid such as nitric acid.It is preferably Methanaminium, N,N,N-trimethyl-, fluoride, described fluorine
Changing reagent and the molar feed ratio of compounds X I is 1~3:1.
The molar feed ratio of described compound V and compounds X I is 1~5:1.
The described response time is detecting that reaction completes.
Step 8:Under inert gas shielding, compound VI and compound VII in polar non-solute,
Under alkalescence condition, carry out substitution reaction, obtain compound VIII.
Described substitution technique and condition can be the conventional method of the such reaction in this area and condition.Preferably,
Above-mentioned preparation method comprises the steps of:Under nitrogen protection, compound VI and compound VII is added to polarity
In aprotic solvent, under room temperature, stirring half an hour all dissolves to raw material, adds alkali and carries out substitution reaction,
Response time is 3~5 hours.After the completion of reaction, frequent pressure-off is molten, and organic solvent is pulled an oar and recrystallization,
Lyophilization obtains compound VIII.
Described polar non-solute is acetonitrile, acetone, DMF or dimethyl sulfoxide
(DMSO), preferably DMF.Described organic solvent and the volume mass of compound VI
Than for 5~20mL/g.
Described alkali is potassium acetate, pyridine, diethylamine, triethylamine, the organic base such as diisopropyl ethyl amine,
It is preferably diisopropyl ethyl amine.The molar feed ratio of described alkali and compound VI is 1~2:1.
The molar feed ratio of described compound VII and compound VI is 1~2:1.
Described making beating use solvent be, oxolane, acetone, dichloromethane, ethyl acetate, methanol,
Toluene, the combination in any of the organic solvent such as petroleum ether or above-mentioned solvent.Preferably dichloromethane and petroleum ether is according to 1:
1 volume ratio mixing.Organic solvent that described making beating uses and the volume mass of compound VI than for 4~
5mL/g.
The solvent that described recrystallization uses is dimethyl sulfoxide, oxolane, acetone, dichloromethane, acetic acid,
Trifluoroacetic acid, ethyl acetate, methanol, toluene, the combination in any of the organic solvent such as petroleum ether or above-mentioned solvent.
Preferably acetic acid methanol is according to 1:1 volume ratio mixing.Organic solvent and compound VI that described recrystallization uses
Volume mass than for 5~20mL/g.
Advantages of the present invention essentially consists in:
1) preparation of connexon VIII, patent US20050238649 and document Bioconjugate
Chem, 2002,13,855-869 all do not mention the physical property of intermediate, but in the middle of finding in experimentation
The easy moisture absorption of body compound IV, this undoubtedly increased the difficulty of post processing.Based on the fact above, the present invention is led to
Cross the method to protection on compound IV hydroxyl, improve hygroscopicity, simplify post-processing operation.
2) post-processing operation of the present invention is simple, by making beating, the method such as recrystallization is multiple batches of obtain pure
Degree>99%, de>98%, single miscellaneous<0.1% finished product ADC connexon VIII, meets pharmaceutical grade crude drug
Require.
3) this synthesising method reacting condition is gentle, and operational approach is simple and convenient, is not only suitable for laboratory and prepares on a small quantity,
It also is adapted for industrialization large-scale production.
4) present invention has good reference meaning for developing new antibody coupling medicine connexon.
Specific embodiment:
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, is generally carried out according to normal condition.
In embodiment, raw material used or reagent are in addition to special instruction, all commercially available.
Room temperature described in embodiment refers both to 20~35 DEG C.Unless otherwise indicated, described reagent is not purified
Directly use.All solvents are purchased from commercialization supplier, such as aldrich (Aldrich), and without place
Reason can use.Reaction is analyzed by TLC and/or is analyzed by LC-MS, by the consumption of parent material
To judge the termination reacted.The thin layer chromatography (TLC) of analysis is in 0.25 millimeter of pre-coated silica gel 60 F254
Carry out on the glass plate (EMD chemical company (EMD Chemicals)) of plate, with UV light (254nm) and/
Or the iodine developing on silica gel, and/or with TLC product dyed thereby such as alcohol phosphomolybdic acid, ninhydrin solution, Gao Meng
Sour potassium solution or ceric sulfate solution heat together.
1H-NMR spectrum is on ten thousand Ruian-Mo Qiuli-VX400 (Varian Mercury-VX400) instrument,
The lower record of 400MHz operation.
Used in the present invention, abbreviation has this area conventional sense, such as:DCM represents dichloromethane, DMF
Represent DMF, PE represents petroleum ether, and EA represents ethyl acetate, and HOAc represents acetic acid,
MeOH represents methanol.
Embodiment 1:(9H- fluorenes -9- base) methyl ((S) -1- (((S) -1- ((4- (((tert-butyl group diformazan
Base silicyl) epoxide) methyl) phenyl) amino) -1- oxo -5- urea groups-amyl- 2- yl) amino) -3-
Methyl isophthalic acid-oxo butyl- 2- yl) carbamic preparation
Under nitrogen protection, by compound I (1.08kg, 2.18mol, 1.0eq) and compound IX-1 (517.6
G, 2.18mol, 1.0eq) it is added in ethyl acetate (20.0L), under room temperature, stirring half an hour is complete to raw material
Portion dissolves, then by ethyl chloroformate (236.6g, 2.18mol, 1.0eq) and triethylamine (220.6g, 2.18
Mol, 1.0eq) ethyl acetate (1.6L) solution be added in reaction system, Deca process temperature control
At -5~-15 DEG C.Completion of dropping, is slowly increased to room temperature and continues reaction up in liquid phase tracing display reaction system
Raw material I disappears, stopped reaction.After reaction stops, frequent pressure-off is molten, and remaining solid is with petroleum ether (1L × 3)
Washing, vacuum drying obtains white solid X-1 (1258.7g, yield:80%, purity:99.2%, de:
99.4%).
1H NMR(400MHz,DMSO-d6) δ 9.98 (s, 1H), 8.07 (d, J=7.2,1H), 7.83 (d, J=7.6,
2H),7.71-7.67(m,2H),7.53-7.51(m,2H),7.43-7.40(m,1H),7.37-7.33(m,2H),
7.28-7.25 (m, 2H), 7.18-7.16 (m, 2H), 6.01 (t, J=5.2,1H), 5.44-5.40 (m, 2H), 4.64 (s,
2H),4.44-4.43(m,1H),4.31-4.23(m,3H),3.96-3.92(m,1H),3.05-2.94(m,2H),
2.01-1.98(m,1H),1.75-1.55(m,2H),1.46-1.37(m,2H),0.88-0.85(m,15H),0.06(s,
6H).LC/MS:[M+H]+=716.
Embodiment 2:(S) -2- ((S) -2- amino -3- methybutane amide groups)-N- (4- (((tertiary fourth
Base dimetylsilyl) epoxide) methyl) phenyl) -5- urea groups pentanamide preparation
Under room temperature, compounds X -1 (3.44kg, 4.8mol, 1.0eq) is added to DMF (17.2
L, in), stirring half an hour all dissolves to raw material, adds diethylamine (700.8g, 9.6mol, 2.0eq),
Until raw material X-1 disappears in liquid phase tracing display reaction system, stopped reaction.After reaction stops, normal pressure precipitation
(spinning off the 3/4 of overall solution volume), then residue is added in ethyl acetate (5L), after stirring to clarify
To in system, Deca petroleum ether occurs to white opacity liquid, more anti-Deca ethyl acetate is to just molten clear, and room temperature opens
Mouth standing 16~48 hours, has solid to separate out, filters, and collects filter cake and is washed with petroleum ether (3L × 2),
Vacuum drying, obtains white solid XI-1 (2045.0g, yield:85%, purity:98.5%, de:98.7%).
1H-NMR(400MHz,DMSO-d6)δ10.08(s,1H),8.14-8.12(m,1H),7.58-7.55(m,
2H),7.24-7.22(m,2H),5.99(s,1H),5.43(s,2H),4.64(s,2H),4.49-4.47(m,1H),
3.02-2.92(m,3H),1.73-1.70(m,1H),1.57-1.59(m,4H),1.41-1.37(m,2H),
0.89-0.88 (m, 12H), 0.78 (d, J=6.8,3H), 0.06 (s, 6H) .LC/MS:[M+H]+=494.
Embodiment 3:6- (2,5- dioxo -2,5- dihydro -1H- pyrroles's -1- base)-N- ((S) -1- (((S)
- 1- ((4- (methylol) phenyl) amino) the amyl- 2- yl of -1- oxo -5- urea groups) amino) -3- methyl isophthalic acid -
Oxo butyl- 2- yl) caproamide preparation
Under room temperature by compounds X I-1 (269g, 0.55mol, 1.0eq) and compound V (168.1g, 0.55
Mol, 1.0eq) it is added to DMF (1L), under room temperature, stirring half an hour all dissolves to raw material,
Then heat to react 4~6 hours at 45~50 DEG C, obtain substituent reactant liquor, reactant liquor is cooled to -5~
0 DEG C of DMF (350mL) adding Methanaminium, N,N,N-trimethyl-, fluoride (50.7g, 0.55mol, 1.0eq)
In solution, completion of dropping, it is warmed to room temperature reaction until raw material disappears in liquid phase tracing display reaction system, stop
Only react.After reaction stops, adding water to separate out to no solid again toward in reactant liquor, filter, collect filter cake.
Filter cake is pulled an oar half an hour with water (400mL) and ethyl acetate (800mL), filters, and collects filter cake, very
Sky is dried to obtain off-white powder VI (292.7g, yield:92%, purity:99.0%, de:98.2%).
1H-NMR(400MHz,DMSO-d6) δ 9.87 (s, 1H), 8.03 (d, J=7.2,1H), 7.78 (d, J=8.8,
1H), 7.49 (d, J=8.8,2H), 7.17 (d, J=8.0,1H), 6.95 (s, 2H), 5.96 (s, 1H), 5.38 (s, 2H),
5.05 (t, J=5.6,1H), 4.37-4.33 (m, 3H), 4.13 (m, 1H), 3.40-3.21 (m, 2H), 2.95-2.89 (m,
2H),2.11-2.07(m,2H),1.92-1.90(m,1H),1.63-1.42(m,9H),1.14-1.12(m,2H),
0.80-0.75(m,6H).LC/MS:[M+H]+=573.
Embodiment 4:4- ((S) -2- ((S) -2- (6- (2,5- dioxo -2,5- dihydro -1H- pyrroles
- 1- base) hexanoyl amido) -3- methylbutyryl ammonia) -5- urea groups amyl group) benzyl (4- nitrobenzophenone) carbonic ester
Preparation
Under nitrogen protection, by compound VI (198.0g, 345.9mmol, 1.0eq) and compound VII (105.2
G, 345.9mmol, 1.0eq) it is added in DMF (990mL), stir under room temperature
Half an hour all dissolves to raw material, adds diisopropyl ethyl amine (44.7g, 345.9mmol, 1.0eq)
Carry out substitution reaction, the response time is 3~5 hours.After the completion of reaction, normal pressure precipitation (spins off overall solution
Long-pending 3/4), residue DCM:PE=1:1 (v/v) (792mL) pulls an oar, and making beating finishes, and filters,
Collect filter cake, then use HOAc:MeOH=1:1 (v/v) (990mL) dissolving filter cake, adds toward in filtrate
Entering water until no longer there being solid to separate out, filtering, collect filter cake and washed with water (500mL × 3), vacuum drying
Obtain white solid VIII (226.2g, yield:88%, purity:99.2%, de:99.3%, single miscellaneous<0.1%).
1H-NMR(400MHz,CDCl3) 8.23 (d, J=8.8,2H), 7.65 (d, J=8.8,2H), 7.42-7.39 (m,
4H), 6.66 (s, 2H), 5.26 (s, 2H), 4.59-4.55 (m, 1H), 4.20-4.16 (m, 1H), 3.48 (t, J=7.2,2H),
3.33-3.17 (m, 2H), 2.29-2.25 (t, J=7.2,2H), 2.01-1.89 (m, 2H), 1.74-1.31 (m, 9H),
0.95-0.90(m,6H).LC/MS:[M+H]+=738.
Embodiment 5:(9H- fluorenes -9- base) methyl ((S) -1- (((S) -1- ((4- (((tert-butyl group diformazan
Base silicyl) epoxide) methyl) phenyl) amino) -1- oxo -5- urea groups-amyl- 2- yl) amino) -3-
Methyl isophthalic acid-oxo butyl- 2- yl) carbamic preparation
Nitrogen protection under, by compound I (540g, 1.09mol, 1.0eq) and compound IX-1 (258.8g,
1.09mol, 1.0eq), it is added in DMF (10.0L), under room temperature, stirring half an hour is all molten to raw material
Solution, then by ethyl chloroformate (470.88g, 4.36mol, 4.0eq) and triethylamine (660.54g, 6.54mol,
DMF (0.8L) solution 6.0eq) is added in reaction system, and Deca process temperature controls at -5~-15 DEG C.
Completion of dropping, is slowly increased to room temperature and continues reaction until raw material I disappears in liquid phase tracing display reaction system, stop
Only react.Reaction adds the water of 4L to separate out to solid after stopping toward in reaction system, filters, collects filter cake,
With water (1L × 2), petroleum ether (1L × 3) washs, drying obtain off-white powder X-1 (550.7g,
Yield:70%, purity:99.2%, de:99.0%).
1H NMR(400MHz,DMSO-d6) δ 9.98 (s, 1H), 8.07 (d, J=7.2,1H), 7.83 (d, J=7.6,
2H),7.71-7.67(m,2H),7.53-7.51(m,2H),7.43-7.40(m,1H),7.37-7.33(m,2H),
7.28-7.25 (m, 2H), 7.18-7.16 (m, 2H), 6.01 (t, J=5.2,1H), 5.44-5.40 (m, 2H), 4.64 (s,
2H),4.44-4.43(m,1H),4.31-4.23(m,3H),3.96-3.92(m,1H),3.05-2.94(m,2H),
2.01-1.98(m,1H),1.75-1.55(m,2H),1.46-1.37(m,2H),0.88-0.85(m,15H),0.06(s,
6H).LC/MS:[M+H]+=716.
Embodiment 6:(S) -2- ((S) -2- amino -3- methybutane amide groups)-N- (4- (((tertiary fourth
Base dimetylsilyl) epoxide) methyl) phenyl) -5- urea groups pentanamide preparation
Under room temperature, compounds X -1 (344g, 0.48mol, 1.0eq) is added to DMF (6.9
L, in), stirring half an hour all dissolves to raw material, adds triethylamine (242.9g, 2.4mol, 5.0eq),
Until raw material X-1 disappears in liquid phase tracing display reaction system, stopped reaction.After reaction stops, normal pressure precipitation
(spinning off the 3/4 of overall solution volume), then residue is added in ethyl acetate (500mL), stirs to clear
In backward clearly system, Deca petroleum ether occurs to white opacity liquid, more anti-Deca ethyl acetate is to just molten clear, room
The uncovered standing of temperature 16~48 hours, has solid to separate out, filters, and collects filter cake with petroleum ether (300mL × 2)
Washing, vacuum drying, obtain white solid XI-1.(172.4g, yield:71%, purity:97.6%, de:
98.0%).
1H-NMR(400MHz,DMSO-d6)δ10.08(s,1H),8.14-8.12(m,1H),7.58-7.55(m,
2H),7.24-7.22(m,2H),5.99(s,1H),5.43(s,2H),4.64(s,2H),4.49-4.47(m,1H),
3.02-2.92(m,3H),1.73-1.70(m,1H),1.57-1.59(m,4H),1.41-1.37(m,2H),
0.89-0.88 (m, 12H), 0.78 (d, J=6.8,3H), 0.06 (s, 6H) .LC/MS:[M+H]+=494.
Embodiment 7:6- (2,5- dioxo -2,5- dihydro -1H- pyrroles's -1- base)-N- ((S) -1- (((S)
- 1- ((4- (methylol) phenyl) amino) the amyl- 2- yl of -1- oxo -5- urea groups) amino) -3- methyl isophthalic acid -
Oxo butyl- 2- yl) caproamide preparation
Under room temperature by compounds X I-1 (538g, 1.09mol, 1.0eq) and compound V (1680.18g, 5.45
Mol, 5.0eq) it is added in acetonitrile (10.7L), under room temperature, stirring half an hour all dissolves to raw material, then
It is warming up to and reacts 4~6 hours at 45~50 DEG C, obtain substituent reactant liquor, reactant liquor is cooled to -5~0 DEG C
Add hydrochloric acid (2M, 1.6L, 3.27mol, 3.0eq) completion of dropping, be warmed to room temperature reaction until liquid phase with
In track display reaction system, raw material disappears, stopped reaction.After reaction stops, reactant liquor is cooled to -5~0 DEG C,
Deca saturated sodium bicarbonate aqueous solution is to pH value 7~8.Completion of dropping, filters, and collects filter cake.Filter cake is used
Water (800mL) and ethyl acetate (1.6L) making beating half an hour, filter, collect filter cake, be vacuum dried
To off-white powder VI (466.1g, yield:74%, purity:99.1%, de:99.6%).
1H-NMR(400MHz,DMSO-d6) δ 9.87 (s, 1H), 8.03 (d, J=7.2,1H), 7.78 (d, J=8.8,
1H), 7.49 (d, J=8.8,2H), 7.17 (d, J=8.0,1H), 6.95 (s, 2H), 5.96 (s, 1H), 5.38 (s, 2H),
5.05 (t, J=5.6,1H), 4.37-4.33 (m, 3H), 4.13 (m, 1H), 3.21-3.40 (m, 2H), 2.95-2.89 (m,
2H),2.11-2.07(m,2H),1.92-1.90(m,1H),1.63-1.42(m,9H),1.14-1.12(m,2H),
0.80-0.75(m,6H).LC/MS:[M+H]+=573.
Embodiment 8:4- ((S) -2- ((S) -2- (6- (2,5- dioxo -2,5- dihydro -1H- pyrroles
- 1- base) hexanoyl amido) -3- methylbutyryl ammonia) -5- urea groups amyl group) benzyl (4- nitrobenzophenone) carbonic ester
Preparation
Under nitrogen protection, by compound VI (1.98Kg, 3.46mol, 1.0eq) and compound VII (1.05
Kg, 3.46mol, 1.0eq) it is added in DMF (39.6L), stirring half under room temperature
Hour all dissolves to raw material, adds diisopropyl ethyl amine (89.4g, 6.92mol, 2.0eq) and carries out
Substitution reaction, the response time is 3~5 hours.After the completion of reaction, normal pressure precipitation (spins off overall solution volume
3/4), residue is pulled an oar with DCM (8L), and making beating finishes, and filters, and collects filter cake, then uses HOAc:
MeOH=1:1 (v/v) (39.6L) dissolving filter cake, adds water until no longer there being solid to separate out toward in filtrate,
Filter, collect filter cake and washed with water (5L × 3), vacuum drying obtains white solid VIII, and (2.14Kg receives
Rate:83%, purity:99.2%, de:99.4%, single miscellaneous<0.1%).
1H-NMR(400MHz,CDCl3) 8.23 (d, J=8.8,2H), 7.65 (d, J=8.8,2H), 7.42-7.39 (m,
4H), 6.66 (s, 2H), 5.26 (s, 2H), 4.59-4.55 (m, 1H), 4.20-4.16 (m, 1H), 3.48 (t, J=7.2,2H),
3.33-3.17 (m, 2H), 2.29-2.25 (t, J=7.2,2H), 2.01-1.89 (m, 2H), 1.74-1.31 (m, 9H),
0.95-0.90(m,6H).LC/MS:[M+H]+=738.
Embodiment 9:(9H- fluorenes -9- base) methyl ((S) -1- (((S) -1- ((4- ((methoxyl group methoxy
Base) methyl) phenyl) amino) the amyl- 2- yl of -1- oxo -5- urea groups) amino) -3- methyl isophthalic acid-oxo butyl- 2-
Base) carbamic preparation
Under nitrogen protection, by compound I (2.7kg, 5.44mol, 1.0eq) and compound IX-2 (3638.4
G, 21.76mol, 4.0eq) it is added in ethyl acetate (13L), under room temperature, stirring half an hour is whole to raw material
Dissolving.Again by DMTMM (6413.5g, 21.76mol, 4.0eq) and potassium carbonate (751.9g, 5.44mol,
Ethyl acetate (0.5L) solution 1.0eq) is added in reactant liquor, and Deca process temperature controls -5~
-15℃.Completion of dropping, is slowly increased to room temperature and continues to react up to raw material I in liquid phase tracing display reaction system
Disappear, stopped reaction.Reaction is washed with petroleum ether (3L × 3) through normal pressure precipitation, remaining solid after stopping,
Vacuum drying obtains off-white powder X-1 (2880.9g, yield:81.6%, purity:99.5%, de:99.2%).
1H NMR (400MHz, DMSO-d6) δ 9.98 (s, 1H), 8.07 (d, J=7.2,1H), 7.83 (d, J=7.6,
2H),7.71-7.67(m,2H),7.53-7.51(m,2H),7.43-7.40(m,1H),7.37-7.33(m,2H),
7.28-7.25 (m, 2H), 7.18-7.16 (m, 2H), 6.01 (t, J=5.2,1H), 5.44-5.40 (m, 2H), 4.64 (s,
2H),4.44-4.43(m,3H),4.31-4.23(m,3H),3.96-3.92(m,1H),3.31(s,3H),
3.05-2.94(m,2H),2.01-1.98(m,1H),1.75-1.55(m,2H),1.46-1.37(m,2H),0.85(d,
J=6.8,6H) .LC/MS:[M+H]+=646.
Embodiment 10:(S) -2- ((S) -2- amino -3- methylbutyrylamino)-N- (4- ((methoxyl group
Methoxyl group) methyl) phenyl) and -5- urea groups pentanamide preparation
Under room temperature, compounds X -2 (310.0g, 0.48mol, 1.0eq) is added in acetonitrile (1.6L),
Stirring half an hour all dissolves to raw material, adds diethylamine (70.1g, 0.96mol, 2.0eq), until
In liquid phase tracing display reaction system, raw material X-2 disappears, stopped reaction.After reaction stops, (the rotation of normal pressure precipitation
Fall the 3/4 of overall solution volume), then residue is added in ethyl acetate (500mL), after stirring to clarify
To in system, Deca petroleum ether occurs to white opacity liquid, more anti-Deca ethyl acetate is to just molten clear, and room temperature opens
Mouth standing 16~48 hours, has solid to separate out, filters, and collects filter cake petroleum ether (300mL × 2) and washes
Wash, vacuum drying, obtain white solid XI-2 (173.9g, yield:84%, purity:98.2%, de:
98.1%).
1H-NMR(400MHz,DMSO-d6)δ10.08(s,1H),8.14-8.12(m,1H),7.58-7.55(m,
2H),7.24-7.22(m,2H),5.99(s,1H),5.23(s,2H),4.64(s,2H),4.49-4.47(m,3H),
3.31(s,3H),3.02-2.92(m,3H),1.73-1.70(m,1H),1.57-1.59(m,4H),1.41-1.37(m,
2H), 0.88 (d, J=6.8,3H), 0.78 (d, J=6.8,3H) .LC/MS:[M+H]+=424.
Embodiment 11:6- (2,5- dioxo -2,5- dihydro -1H- pyrroles's -1- base)-N- ((S) -1-
(((S) -1- ((4- (methylol) phenyl) amino) the amyl- 2- yl of -1- oxo -5- urea groups) amino) -3-
Methyl isophthalic acid-oxo butyl- 2- yl) caproamide preparation
Under room temperature by compounds X I-2 (269.0g, 635.2mmol, 1.0eq) and compound V (195.8g,
635.2mmol, 1.0eq) it is dissolved in DMF (5.38L), stir half an hour extremely under room temperature
Raw material all dissolves, and then heats to react 4~6 hours at 45~50 DEG C, obtains substituent reactant liquor, will
Reactant liquor be cooled to -5~0 DEG C add trifluoroacetic acid (217.2g, 1905.6mmol, 3.0eq), completion of dropping,
It is warmed to room temperature reaction until raw material disappears in liquid phase tracing display reaction system, stopped reaction.After reaction stops,
Reactant liquor is cooled to -5~0 DEG C, and Deca saturated sodium bicarbonate aqueous solution is to pH value 7~8.Completion of dropping, mistake
Filter, collects filter cake.Filter cake is pulled an oar half an hour with water (400mL) and ethyl acetate (800mL), filters,
Collect filter cake, vacuum drying obtains off-white powder VI (310.7g, yield:85%, purity:99.5%,
de:99.3%).
1H-NMR(400MHz,DMSO-d6) δ 9.87 (s, 1H), 8.03 (d, J=7.2,1H), 7.78 (d, J=8.8,
1H), 7.49 (d, J=8.8,2H), 7.17 (d, J=8.0,1H), 6.95 (s, 2H), 5.96 (s, 1H), 5.38 (s, 2H),
5.05 (t, J=5.6,1H), 4.37-4.33 (m, 3H), 4.13 (m, 1H), 3.40-3.21 (m, 2H), 2.95-2.89 (m,
2H),2.11-2.07(m,2H),1.92-1.90(m,1H),1.63-1.42(m,9H),1.14-1.12(m,2H),
0.80-0.75(m,6H).LC/MS:[M+H]+=573.
Embodiment 12:4- ((S) -2- ((S) -2- (6- (2,5- dioxo -2,5- dihydro -1H- pyrrole
Cough up -1- base) hexanoyl amido) -3- methylbutyryl ammonia) -5- urea groups amyl group) benzyl (4- nitrobenzophenone) carbonic acid
The preparation of ester
Nitrogen protection under, by compound VI (5g, 8.73mmol, 1.0eq) and compound VII (5.3g,
17.46mmol, 2.0eq) it is added in DMF (100mL), under room temperature, stirring half is little
All dissolve up to raw material, add triethylamine (1.77g, 17.46mmol, 2.0eq) and carry out substitution reaction,
Response time is 3~5 hours.After the completion of reaction, normal pressure precipitation (spins off the 3/4 of overall solution volume), remaining
Thing DCM:PE=1:1 (v/v) (25mL) pulls an oar, and making beating finishes, and filters, and collects filter cake, then uses
HOAc:MeOH=4:1 (v/v) (100mL) dissolving filter cake, adds water until no longer having solid toward in filtrate
Body separates out, and filters, and collects filter cake and is washed with water (15mL × 3), vacuum drying obtains white solid VIII
(4.85g, yield:75%, purity:99.7%, de:99.6%, single miscellaneous<0.1%).
1H-NMR(400MHz,CDCl3) 8.23 (d, J=8.8,2H), 7.65 (d, J=8.8,2H), 7.42-7.39 (m,
4H), 6.66 (s, 2H), 5.26 (s, 2H), 4.59-4.55 (m, 1H), 4.20-4.16 (m, 1H), 3.48 (t, J=7.2,2H),
3.33-3.17 (m, 2H), 2.29-2.25 (t, J=7.2,2H), 2.01-1.89 (m, 2H), 1.74-1.31 (m, 9H),
0.95-0.90(m,6H).LC/MS:[M+H]+=738.
The all documents referring in the present invention are all incorporated as reference in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.