CN103387602B - A kind of novel open chain tetrapeptide analogs and its preparation method and application - Google Patents
A kind of novel open chain tetrapeptide analogs and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to the preparation methods and purposes of the open chain tetrapeptide analogs shown in a kind of general formula I, the pharmaceutical composition comprising the open chain tetrapeptide analogs and the open chain tetrapeptide analogs.Compound of the present invention can preferably inhibit Grb2 SH2 active, therefore available for preparing prevention or treatment and the drug of Grb2 SH2 activity and/or the relevant disease of signal.
Description
Technical field
The present invention relates to a kind of novel open chain tetrapeptide analogs, the pharmaceutical composition comprising the open chain tetrapeptide analogs and it is somebody's turn to do
The preparation method and purposes of open chain tetrapeptide analogs, such compound can be used as Grb2-SH2 inhibitor.
Background technology
In recent years, antitumor drug research is from traditional cell toxicity medicament to point for tumorigenesis mechanism
Sub- target spot and molecular targeted agents etc. development.Signal transduction pathway is that cell makes environmental stimuli must way to be reacted
Diameter plays crucial regulating and controlling effect, the close phase of generation, evolution with tumour in the proliferation of cell, differentiation and migration
It closes.The blocking tumour cell signal transduction pathway of selectivity, destroys its automatic control growth regulating mechanism, it has also become very attractive
Antitumor drug research hotspot.
The study found that there are the mutation of Ras genes activation and the increasings of Ras protein expression levels for about 30% human tumor
The phenomenon that high, therefore, the transduction pathway for regulating and controlling Ras signals, are designed in the antitumor drug using intracellular signal transduction pathway as target spot
In play an important role [Songyang, Z., et al.Mol.Cell.Biol.1994,14,2777.].Currently for Ras albumen
The inhibitor of upstream passages has following 3 class：A, Grb2-SH2 inhibitor；B, tyrosine kinase inhibitor (EGFR inhibitor)；C、
Brc Abl inhibitor.Regulation and control Ras signal transduction pathways occupy an important position in treatment of cancer, adjust its upstream related enzyme systems,
It can avoid the activation of Ras albumen；Exploitation blocks the antitumor inhibitor of Ras accesses to become research heat by target spot of Grb2-SH2
Point.
Researcher obtains a kind of non-phosphoric acid Grb2-SH2 cyclic peptide inhibitor G1TE and its class by phage library displaying
Like object.Due to pTyr be Grb2-SH2 native ligands obtain high activity Key residues, and without phosphoric acid cyclic peptide ligand G1TE with
Effective combine of Grb2-SH2 relies primarily on essential amino acid side chain and its specific conformation in peptide chain, therefore, G1TE tools
There is very high selectivity, Grb2-SH2 functional domains can be selectively applied to.On the other hand, G1TE analogs do not contain rich charge
Phosphate group, be conducive to bioactive molecule through cell membrane.Researcher synthesizes many using cyclic peptide G1TE as lead compound
Derivative so that the combination activity of this kind of compound molecule level increases substantially, but the Grb2- with whole cell activity
SH2 is non-, and phosphoric acid cyclic peptide inhibitor is seldom, and these high activity molecules are still 9 peptide structures, and molecular weight is too big, and druggability is not
It is preferable.
The present invention be exactly for disadvantages mentioned above, from G1TE Macrocyclic peptides design features, by contracting ring, open chain, with
The amino acid substitution of structure and function double action has obtained the open chain tetrapeptide analogs of a series of novel.And find them not
It is only high with Grb2-SH2 affinities on a molecular scale, and with preferable cell activity, there is good development prospect.
Invention content
Goal of the invention
The object of the present invention is to provide a kind of general formula I represent open chain tetrapeptide analogs its isomers or its pharmaceutically
Acceptable salt, ester, prodrug or hydrate.
It is a further object of the present invention to provide the preparation methods of the general formula I compounds represented.
Another object of the present invention is to provide the open chain tetrapeptide analogs that the general formula I represents or its isomers or its pharmacy
The upper purposes of acceptable salt, ester, prodrug or hydrate in Grb2-SH2 inhibitor is prepared, the inhibitor are used to treat
And/or prevent and pathogenicity or undesirable Grb2-SH2 activity and/or the relevant disease of signal.
It is yet another object of the invention to provide it is a kind of comprising the present invention general formula I represent compound its isomers or its
Pharmaceutically acceptable salt, ester, prodrug or hydrate pharmaceutical composition.
Technical solution
To achieve these goals, the present invention provides the open chain tetrapeptide analogs or its isomers that below formula I is represented
Or its pharmaceutically acceptable salt, ester, prodrug or hydrate,
Wherein, R1For hydrogen, 9- fluorenes methoxycarbonyl, C6-C10Aryl carbonyl, C5-C10Aromatic heterocycle base carbonyl, C6-C10
The C of aryl substitution1-C6Alkoxy carbonyl, C5-C10The C of aromatic heterocycle base substitution1-C6Alkoxy carbonyl, C6-C10Aryl replaces
C1-C6Alkyl-carbonyl or C5-C10The C of aromatic heterocycle base substitution1-C6Alkyl-carbonyl, wherein, the C5-C10Aromatic heterocycle
Base contains the 1-3 hetero atom in N, O and S, the C6-C10Aryl or C5-C10Aromatic heterocycle base can be not necessarily
By selected from halogen, hydroxyl, amino, nitro, trifluoromethyl, carboxyl ,-CN, C1-C6Alkyl, C1-C6Alkoxy, 9- fluorenes methoxyl group acyls
Amido, C1-C6Alkyl amino and C6-C101-3 substituent group in aryloxy group is replaced；R1Preferably hydrogen, 9- fluorenes methoxyl group carbonyls
Base, C6-C10Aryl carbonyl, C6-C10The C of aryl substitution1-C6Alkyloxycarbonyl or C6-C10The C of aryl substitution1-C6Alkyl-carbonyl,
The C6-C10Aryl is not necessarily by selected from halogen, hydroxyl, amino, nitro, trifluoromethyl, carboxyl ,-CN, C1-C6Alkyl, C1-
C6Alkoxy, 9- fluorenes methoxyamide bases and C1-C61-3 substituent group in alkyl amino is replaced；R1More preferably hydrogen, 2-
Amino benzoyl group, 3- amino benzoyl group, 4- amino benzoyl group, 3- aminobenzoics Epoxide carbonyl, 2- aminobenzoics Epoxide carbonyl, 4-
Aminobenzoic Epoxide carbonyl, 2- (9- fluorenes methoxyamides base) benzoyl group, 3- (9- fluorenes methoxyamides base) benzoyl group, 4- (9-
Fluorenes methoxyamide base) benzoyl group or 9- fluorenes methoxycarbonyls；
R2And R3Can be identical or different, it is each independently hydrogen, C1-C6Alkyl, C2-C6Alkenyl, C1-C5Alkylidene carboxylic
Base, C1-C5The double carboxyls of alkylidene,OrWherein, 0≤n
≤6；R2And R3It is preferably hydrogen, methyl, ethyl, propyl, isopropyl, normal-butyl, tertiary butyl, isobutyl group ,-CH each independently2CH
(COOH)2Or-(CH2)3-COOH；
R4For C1-C6Alkyl, C2-C6Alkenyl, C6-C10Aryl, C5-C10Aromatic heterocycle base, C6-C10Aryl substitution
C1-C6Alkyl, C5-C10The C of aromatic heterocycle base substitution1-C6Alkyl or C4-C7The C of saturated heterocyclyl substitution1-C6Alkyl, wherein,
The C4-C7Saturated heterocyclyl or C5-C10Aromatic heterocycle base contains the 1-3 hetero atom in N, O and S, the C6-C10
Aryl or C5-C10Aromatic heterocycle base can not necessarily by selected from halogen, hydroxyl, amino, nitro, trifluoromethyl, carboxyl ,-
CN、C1-C6Alkyl, C1-C6Acyl group, C1-C6Alkoxy, C1-C6Alkyl amino and C6-C101-3 substituent group in aryloxy group is taken
Generation；R4Preferably C1-C6Alkyl, C2-C6It is alkenyl, unsubstituted or by selected from C1-C6Alkyl, hydroxyl, amino and trifluoromethyl
At least one of substituent group substitution benzyl；R4More preferably methyl, ethyl, propyl, isopropyl, normal-butyl, tertiary butyl, different
Butyl, benzyl, 2- methylbenzyls, 3- methylbenzyls, 4- methylbenzyls or 3- (amino) 4- (hydroxyl) benzyl.
In the present invention, particularly preferred particular compound is compound prepared by the embodiment of the present invention.Specially：
1 compound 2 of compound
LSG-100 LSG-101
3 compound 4 of compound
LSG-102 LSG-103
5 compound 6 of compound
LSG-104 LSG-105
7 compound 8 of compound
LSG-106 LSG-110
9 compound 10 of compound
LSG-112 LSG-113
11 compound 12 of compound
LSG-114 LSG-120
13 compound 14 of compound
LSG-89 LSG-90
15 compound 16 of compound
LSG-91 LSG-93
Compound 17
LSG-94
Another technical solution of the present invention provides the preparation method of the compounds of this invention, and this method can be shown in following
It is prepared by flow：
Generalized flowsheet
Wherein R1、R2、R3And R4It is as defined above；
Step a)：Rink resins or PAL resins and Nα(9- fluorenes methoxycarbonyl)-NγTrityl-L- asparagus fern acyls
Amine (Fmoc-Asn (Trt)-OH) is dissolved in N,N-dimethylformamide (DMF) in benzotriazole-N, N, N ', N '-tetramethyl
Compound II is obtained by the reaction under urea hexafluorophosphate (HBTU) and N, N- diisopropylethylamine (DIPEA) effect and in microwave；
Step b)：Compound II is dissolved in 20% piperidines/n,N-Dimethylformamide (DMF) (v/v), in deprotection agent
【20% piperidines/n,N-Dimethylformamide (DMF) (v/v), 0.1N I-hydroxybenzotriazoles (HOBT)】Under the action of in microwave
In compound III is obtained by the reaction；
Step c)：Step a and b are repeated by raw material of compound III, the connection of its excess-three amino acid is obtained into chemical combination up
Object IV；
Step d)：By decomposition agent (trifluoroacetic acid/triethylsilane/water (TFA/TES/H2O)：7.6:0.2:0.2 (volume
Than)) be added in compound IV, obtain compound V after shaking 2 hours at room temperature.
Another technical solution of the present invention provides the purposes for the compound that general formula I of the present invention is represented, is preparing
Purposes in Grb2-SH2 inhibitor, the Grb2-SH2 inhibitor is for treating or preventing and pathogenicity or undesirable
Grb2-SH2 activity and/or the relevant disease of signal are being prepared for treatment or prevention as caused by the activation of Grb2-SH2
Purposes in the drug of disease.Specifically, the disease includes but is not limited to cancer, for example, breast cancer, oophoroma or
Solid tumor, such as brain tumor, prostate tumor and leukaemia, including chronic myelogenous leukemia, lymthoma, autoimmune disease,
Inflammation, metabolic disease, diabetes, obesity and cardiovascular and cerebrovascular disease etc..
In the fourth aspect of the present invention, the change that a kind of one or more general formula I comprising therapeutically effective amount are represented is provided
Close the pharmaceutical composition of object or its isomers or its pharmaceutically acceptable salt, ester, prodrug or hydrate, the composition can be into
One step includes the excipient allowed on a certain amount of galenic pharmacy.
According to another aspect of the invention, the side for treating or preventing the disease as caused by the activation of Grb2-SH2 is provided
Method, the method includes the compounds that represent of general formula I of application therapeutically effective amount or its isomers or its is pharmaceutically acceptable
Salt, ester, prodrug or hydrate are to patient.
Advantageous effect
Open chain tetrapeptide analogs provided by the invention have the following advantages：
1st, the compound that general formula I is represented can effectively inhibit Grb2-SH2 in range of doses；
2nd, the pharmaceutical composition for the compound that general formula I containing therapeutically effective amount is represented can effectively treat cancer, white
Blood disease, autoimmune disease and cardiovascular and cerebrovascular disease etc.；
3rd, the pharmaceutical composition of excipient allowed in the compound and galenic pharmacy that the general formula I comprising therapeutically effective amount is represented
The effect in above-mentioned 1 and 2 can equally be played.
Description of the drawings
Fig. 1 is the schematic diagram for the intracellular reactive for showing the compounds of this invention 12, dense in difference for performance compound 12
To the growth inhibiting schematic diagrames of breast cancer cell MDA-MB-453 under degree.
Specific embodiment
With reference to specific embodiment, the invention will be further described.It should be understood that these embodiments are merely to illustrate this
It invents and does not limit the scope of the invention.
Laboratory apparatus
Microwave Peptide synthesizer model (Microwave Peptide Synthesizer, CEM Liberty 1), matter
EI-MS 95 mass spectrographs of Finnigan MAT are composed, ESI-MS is measured using Finnigan LCQ Deca mass spectrographs.Compound
Purity testing using two solvent system high performance liquid chromatography (HPLC, Waters1525 2489 2707) carry out Purity analysis
(system 1：Solvent forms A：0.05%TFA aqueous solutions；B：0.05%TFA acetonitrile solutions.System 2：Solvent forms：A：0.05%TFA
Aqueous solution；C：0.05%TFA methanol solutions).Preparing column type number is：Vydac C18,120A(10mmID×250mm)；
XBridgeTMC18,5 μM, ultraviolet detection at 19 × 150mm, 225nm or 210nm；Analytical column model：SunfireTM,C18,
3.5 μM, ultraviolet detection at 4.6mm × 150mm, 225nm or 210nm.
Prepare embodiment
Embodiment 1
Compound 1：LSG-100
Synthesis step：
1st, resin swelling：PAL resins (217mg) (200-400 mesh, carrying capacity 0.46mmol/g, BACHEM, D-2125) are put
In microwave reactor, n,N-Dimethylformamide (5ml) and dichloromethane (5ml) are added in, after 900 seconds, with N, N- dimethyl
Formamide elutes (7ml × 4 time).
2nd, (S) -2,4- diamino -4- oxobutanoyls are connect (asparagine (Asn) being protected with amino reacts)：
N is added in resin after swellingα(9- fluorenes methoxycarbonyl)-NγTrityl-altheine (Fmoc-Asn (Trt)-
OH) (2.5ml, 0.5mmol), benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate (HBTU) (1ml,
0.5mmol), n,N-diisopropylethylamine (0.5ml, 1mmol), microwave power set 27W, temperature are 75 DEG C, the time 300
Second.After having reacted, eluted (7ml × 4 time) with n,N-Dimethylformamide.
3rd, 1- aminocyclohexyls formoxyl (Ac6c) is connect：In the compound obtained in step 2 add in [20% piperidines N, N- bis-
Methylformamide (v/v), 0.1N I-hydroxybenzotriazoles (HOBT)] (7ml), microwave reaction, initially deprotect (37W, 75 DEG C,
30S), after having reacted, with DMF (7ml) elute, add [20% piperidines n,N-Dimethylformamide (v/v), 0.1N 1- hydroxyls
Benzotriazole] (7ml), microwave reaction, deprotection (37W, 75 DEG C, 180S).After having reacted, again with n,N-Dimethylformamide
It elutes (7ml × 4 time), adds in 1- (N- (9- fluorenes methoxycarbonyl)-amino)-hexahydrobenzoid acid (Fmoc-Ac6c-OH)
(2.5ml, 0.5mmol), benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate (1ml, 0.5mmol), N, N- bis- are different
Propylethylamine (0.5ml, 1mmol), microwave power set 27W, after temperature is 75 DEG C, 300 seconds, n,N-Dimethylformamide leaching
It washes (7ml × 4 time).
4th, connect isobutylamino acetyl group (leucine (Leu) being protected with amino reacts)：In the change that step 3 obtains
It closes and [20% piperidines n,N-Dimethylformamide (v/v), 0.1N I-hydroxybenzotriazoles] (7ml) is added in object, microwave reaction,
Initial deprotection (37W, 75 DEG C, 30S), after react, with n,N-Dimethylformamide (7ml) elution, add [20% piperidines
N,N-Dimethylformamide (v/v), 0.1N I-hydroxybenzotriazoles] (7ml), microwave reaction, deprotection (37W, 75 DEG C,
180S).It after having reacted, is eluted (7ml × 4 time) with n,N-Dimethylformamide again, adds in N- (9- fluorenes methoxycarbonyl)-L-
Leucine (Fmoc-Leu-OH) (2.5ml, 0.5mmol), benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate
(1ml, 0.5mmol), n,N-diisopropylethylamine (0.5ml, 1mmol), microwave power set 27W, temperature be 75 DEG C, 300
After second, n,N-Dimethylformamide elution (7ml × 4 time).
5th, (S) -2- amino -5- carboxyl -2- methyl-pentanoyl ((S) -2- ammonia being protected with amino and 5- carboxyls is connect
Base -2- methyl adipic acid reacts) ((S)-α-Me-Adi)：In the compound obtained in step 4 add in [20% piperidines N, N- diformazans
Base formamide (v/v), 0.1N I-hydroxybenzotriazoles] (7ml), microwave reaction, initially deprotect (37W, 75 DEG C, 30S), instead
After having answered, with n,N-Dimethylformamide (7ml) elute, add [20% piperidines n,N-Dimethylformamide (v/v), 0.1N
I-hydroxybenzotriazole] (7ml), microwave reaction, deprotection (37W, 75 DEG C, 180S).After having reacted, again with N, N- dimethyl
Formamide elutes (7ml × 4 time), adds in (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxycarbonyl -2- first
Base-valeric acid ((S)-Fmoc- α-Me-Adi (tBu)-OH) (2.5ml, 0.5mmol), benzotriazole-N, N, N ', N '-tetramethyl
Urea hexafluorophosphate (1ml, 0.5mmol), n,N-diisopropylethylamine (0.5ml, 1mmol), microwave power set 27W, temperature
Spend be 75 DEG C, 300 seconds after, n,N-Dimethylformamide elution (7ml × 4 time).
6th, 2- amino benzoyls (Abz) are connect：In the compound obtained in step 5 add in [20% piperidines N, N- dimethyl
Formamide (v/v), 0.1N I-hydroxybenzotriazoles HOBT] (7ml), microwave reaction, initially deprotect (37W, 75 DEG C, 30S),
After having reacted, with n,N-Dimethylformamide (7ml) elute, add [20% piperidines n,N-Dimethylformamide (v/v),
0.1N I-hydroxybenzotriazoles] (7ml), microwave reaction, deprotection (37W, 75 DEG C, 180S).After having reacted, again with N, N-
Dimethylformamide elutes (7ml × 4 time), adds in 2- (N- (9- fluorenes methoxycarbonyl)-amino) benzoic acid (Fmoc-Abz-OH)
(2.5ml, 0.5mmol), benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate (1ml, 0.5mmol), N, N- bis- are different
Propylethylamine (0.5ml, 1mmol), microwave power set 27W, after temperature is 75 DEG C, 300 seconds, n,N-Dimethylformamide leaching
It washes (7ml × 4 time).
7th, it deprotects：In the compound obtained in step 6 add in [20% piperidines n,N-Dimethylformamide (v/v), 0.1N
I-hydroxybenzotriazole] (7ml), microwave reaction, initially deprotect (37W, 75 DEG C, 30S), after having reacted, with N, N- dimethyl
Formamide (7ml) elute, add [20% piperidines n,N-Dimethylformamide (v/v), 0.1N I-hydroxybenzotriazoles
(HOBT)] (7ml), microwave reaction, deprotection (37W, 75 DEG C, 180S).After having reacted, drenched again with n,N-Dimethylformamide
It washes (7ml × 4 time).More than 7 steps reaction be all that microwave Peptide synthesizer is automatically performed, always take time about 2 hours.
8th, the preparation before cracking：Resin is successively with n,N-Dimethylformamide (2ml × 6), dichloromethane (2ml × 6), first
Alcohol (2ml × 6), dichloromethane (2ml × 6) cleaning, the resin vacuum drying after cleaning.
9th, it cracks：By lytic reagent (trifluoroacetic acid/triethylsilane/water：7.6/0.2/0.2) it is added to dry resin
In, it shakes 2 hours at room temperature.Filtering collects filtrate, resin, merging filtrate is cleaned with a small amount of TFA.Filtrate is blown under a nitrogen
It is dry to be concentrated to 1-2ml.
10th, it centrifuges：25ml cold ethers are added in polypeptide concentrate, there are a large amount of solids to be precipitated.Centrifugation 8 minutes
Supernatant is carefully poured out after (3000r/s), adds cold ether, repeatedly this step 3 time, just obtains polypeptide crude product.
11st, it purifies：Use reversed-phased high performace liquid chromatographic purified polypeptide.Liquid-phase condition：Solvent system：0.05% trifluoro second
The aqueous solution of acid；B：The acetonitrile solution of 0.05% trifluoroacetic acid.
12nd, it is freeze-dried.
13rd, structural confirmation：ESI-MS carries out structural confirmation.
ESI-MS m/z：Calculated value 645.4, measured value 646.1 (M+H)+；HPLC purity assays, sample retention time tR=
14.173min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 100.00%)；Or tR=
23.701min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 97.93%).
Prepare embodiment 2
Compound 2：LSG-101
In addition to replacing 2- (N- (9- fluorenes methoxyl groups with 3- (N- (9- fluorenes methoxycarbonyl)-amino)-phenylacetic acid in step 6
Carbonyl)-amino) except-benzoic acid (Fmoc-Abz-OH), implement the step identical with preparation embodiment 1 and obtain title compound
For white powdery solids.
ESI-MS m/z：Calculated value 675.4, measured value 676.5 (M+H)+；HPLC purity assays, sample retention time tR=
15.093min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 96.07%)；Or tR=
23.109min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.00%).
Prepare embodiment 3
Compound 3：LSG-102
Other than saving step 6 and directly being deprotected, the implementation step identical with preparing embodiment 1 obtains titled
Conjunction object is white powdery solids.
ESI-MS m/z：Calculated value 526.3, measured value 527.1 (M+H)+；HPLC purity assays, sample retention time tR=
12.529min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 100.00%)；Or tR=
18.154min (ratio of solvent methanol and water increases to 90%, 99.00% in 30min from 10%).
Prepare embodiment 4
Compound 4：LSG-103
In addition to using (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- two (tert-butoxycarbonyl) -2- in steps of 5
Metliyl-butyric acid ((S)-Fmoc-Gla (tBu) 2-OH) replaces (S)-Fmoc- α-Me-Adi (tBu)-OH, and in step 6 3-
(N- (9- fluorenes methoxycarbonyl)-amino)-phenylacetic acid is replaced except Fmoc-Abz-OH, implements the step identical with preparing embodiment 1
The rapid title compound that obtains is white powdery solids.
ESI-MS m/z：Calculated value 691.3, measured value 692.4 (M+H)+；HPLC purity assays, sample retention time tR=
15.173min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 98.04%)；Or tR=
25.136min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.08%).
Prepare embodiment 5
Compound 5：LSG-104
In addition in step 5 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- two (tert-butoxycarbonyl) -2- first
Base-butyric acid ((S)-Fmoc-Gla (tBu)2- OH) replace (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxy carbonyls
Base -2- methvl-pentanoic acids ((S)-Fmoc- α-Me-Adi (tBu)-OH), and except omitting step 6 and directly being deprotected,
Implement the step identical with preparing embodiment 1 and obtain title compound as white powdery solids.
ESI-MS m/z：Calculated value 542.3, measured value 543.4 (M+H)+；HPLC purity assays, sample retention time tR=
12.133min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 97.00%)；Or tR=
20.121min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 99.05%).
Prepare embodiment 6
Compound 6：LSG-105
In addition in step 5 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- two (tert-butoxycarbonyl) -2- first
Base-butyric acid ((S)-Fmoc-Gla (tBu)2- OH) replace (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxy carbonyls
Except base -2- methvl-pentanoic acids ((S)-Fmoc- α-Me-Adi (tBu)-OH), implement the step identical with preparing embodiment 1 and obtain
Title compound is white powdery solids.
ESI-MS m/z：Calculated value 661.3, measured value 662.1 (M+H)+；HPLC purity assays, sample retention time tR=
16.173min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 96.19%)；Or tR=
23.321min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 97.00%).
Prepare embodiment 7
Compound 7：LSG-106
In addition in step 5 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- two (tert-butoxycarbonyl) -2- first
Base-butyric acid ((S)-Fmoc-Gla (tBu)2- OH) replace (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxy carbonyls
Base -2- methvl-pentanoic acids ((S)-Fmoc- α-Me-Adi (tBu)-OH), and omit step 6 and do not remove amino protecting group and straight
It connects and cuts except resin, implement the step identical with preparing embodiment 1 and obtain title compound as white powdery solids.
ESI-MS m/z：Calculated value 764.3, measured value 765.4 (M+H)+；HPLC purity assays, sample retention time tR=
16.173min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 96.76%)；Or tR=
21.604min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 99.00%).
Prepare embodiment 8
Compound 8：LSG-110
It directly cuts resin in addition to omitting step 6 and not removing amino protecting group and implements the step identical with preparing embodiment 1
Suddenly, title compound is obtained as white powdery solids.
ESI-MS m/z：Calculated value 748.4, measured value 749.5 (M+H)+；HPLC purity assays, sample retention time tR=
17.573min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 97.84%)；Or tR=
24.981min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 95.56%).
Prepare embodiment 9
Compound 9：LSG-112
In addition in step 4 with N- (9- fluorenes methoxycarbonyl)-L-phenylalanines (Fmoc-Phe-OH) instead of N- (9- fluorenes first
Epoxide carbonyl)-L-Leu (Fmoc-Leu-OH), and omit except step 6 directly deprotection, obtaining title compound is
White powdery solids.
ESI-MS m/z：Calculated value 560.3, measured value 561.4 (M+H)+；HPLC purity assays, sample retention time tR=
14.654min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 96.10%)；Or tR=
22.016min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 96.08%).
Prepare embodiment 10
Compound 10：LSG-113
In addition in step 4 with N- (9- fluorenes methoxycarbonyl)-L-4- methylphenylalanines (Fmoc- (4-Me)-Phe-OH)
Instead of N- (9- fluorenes methoxycarbonyl)-L-Leu (Fmoc-Leu-OH), and omit step 6 and do not remove amino protecting group
And directly cut except resin, implement the step identical with preparing embodiment 1 and obtain title compound as white powdery solids.
ESI-MS m/z：Calculated value 796.4, measured value 797.5 (M+H)+；HPLC purity assays, sample retention time tR=
23.463min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 98.36%)；Or tR=
28.473min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 97.81%).
Prepare embodiment 11
Compound 11：LSG-114
In addition in step 4 with N- (9- fluorenes methoxycarbonyl)-L-4- methylphenylalanines (Fmoc- (4-Me)-Phe-OH)
Except N- (9- fluorenes methoxycarbonyl)-L-Leu (Fmoc-Leu-OH), implement the step identical with preparing embodiment 1
Title compound is obtained as white powdery solids.
ESI-MS m/z：Calculated value 693.4, measured value 694.4 (M+H)+；HPLC purity assays, sample retention time tR=
18.400min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 98.68%)；Or tR=
28.143min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 95.11%).
Prepare embodiment 12
Compound 12：LSG-120
In addition in step 4 with N- (9- fluorenes methoxycarbonyl-L-phenylalanine) (Fmoc-Phe-OH) instead of N- (9- fluorenes first
Epoxide carbonyl)-L-Leu (Fmoc-Leu-OH), and omit step 6 and do not remove amino protecting group and directly cut resin it
Outside, implement the step identical with preparing embodiment 1 and obtain title compound as white powdery solids.
ESI-MS m/z：Calculated value 782.4, measured value 783.5 (M+H)+；HPLC purity assays, sample retention time tR=
14.862min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 100.00%)；Or tR=
26.881min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 100.00%).
Prepare embodiment 13
Compound 13：LSG-89
In addition in step 4 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -3- (3- (tritylamino) -4- (three
Benzyloxy) phenyl) propionic acid (Fmoc-3- (NHTrt)-Tyr (Trt)-OH) is instead of N- (9- fluorenes methoxycarbonyl)-L-Leu
(Fmoc-Leu-OH), in step 5 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- bis- (tert-butoxycarbonyl) -2-
Metliyl-butyric acid ((S)-Fmoc-Gla (tBu)2- OH) replace (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxies
Carbonyl -2- methvl-pentanoic acids ((S)-Fmoc- α-Me-Adi (tBu)-OH), and omit step 6 and do not remove amino protecting group and
It directly cuts except resin, implements the step identical with preparing embodiment 1 and obtain title compound as white powdery solids.
ESI-MS m/z：Calculated value 829.3, measured value 830.1 (M+H)+；HPLC purity assays, sample retention time tR=
9.825min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 98.08%)；Or tR=19.143min
(ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.00%).
Prepare embodiment 14
Compound 14：LSG-90
In addition in step 4 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -3- (3- (tritylamino) -4- (three
Benzyloxy) phenyl) propionic acid (Fmoc-3- (NHTrt)-Tyr (Trt)-OH) is instead of N- (9- fluorenes methoxycarbonyl)-L-Leu
(Fmoc-Leu-OH), it and omits step 6 and does not remove amino protecting group and directly cut except resin, implement to implement with preparing
The identical step of example 1 obtains title compound as white powdery solids.
ESI-MS m/z：Calculated value 813.4, measured value 814.1 (M+H)+；HPLC purity assays, sample retention time tR=
15.173min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 98.00%)；Or tR=
24.149min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.08%).
Prepare embodiment 15
Compound 15：LSG-91
In addition in step 4 (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -3- (3- (tritylamino) -4- (three
Benzyloxy) phenyl) propionic acid (Fmoc-3- (NHTrt)-Tyr (Trt)-OH) is instead of N- (9- fluorenes methoxycarbonyl)-L-Leu
(Fmoc-Leu-OH), and step 7 is omitted without in addition to the protecting group to deaminize, implementing the step identical with preparing embodiment 1
The rapid title compound that obtains is white powdery solids.
ESI-MS m/z：Calculated value 932.4, measured value 933.2 (M+H)+；HPLC purity assays, sample retention time tR=
17.143min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 97.60%)；Or tR=
24.431min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.08).
Prepare embodiment 16
Compound 16：LSG-93
In addition in step 4 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -3- (3- (tritylamino) -4- (three
Benzyloxy) phenyl) propionic acid (Fmoc-3- (NHTrt)-Tyr (Trt)-OH) is instead of N- (9- fluorenes methoxycarbonyl)-L-Leu
(Fmoc-Leu-OH), in step 5 (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -4,4- bis- (tert-butoxycarbonyl) -2-
Metliyl-butyric acid ((S)-Fmoc-Gla (tBu)2- OH) replace (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -5- tert-butoxies
Carbonyl -2- methvl-pentanoic acids ((S)-Fmoc- α-Me-Adi (tBu)-OH), and in step 6 3- (N- (9- fluorenes methoxyl group carbonyls
Base)-amino)-phenylacetic acid replace N- (9- fluorenes methoxycarbonyl) -2- aminobenzoic acids (Fmoc-Abz-OH) except, implement and system
The standby identical step of embodiment 1 obtains title compound as white powdery solids.
ESI-MS m/z：Calculated value 756.3, measured value 757.4 (M+H)+；HPLC purity assays, sample retention time tR=
13.143min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 199.03%)；Or tR=
19.231min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 98.88%).
Prepare embodiment 17
Compound 17：LSG-94
In addition in step 4 with (S) -2- (N- (9- fluorenes methoxycarbonyl) amino) -3- (3- (tritylamino) -4- (three
Benzyloxy) phenyl) propionic acid (Fmoc-3- (NHTrt)-Tyr (Trt)-OH) is instead of N- (9- fluorenes methoxycarbonyl)-L-Leu
(Fmoc-Leu-OH), N- (9- fluorenes methoxyl group carbonyls are replaced with 3- (N- (9- fluorenes methoxycarbonyl)-amino)-phenylacetic acid in step 6
Base) except -2- aminobenzoic acids (Fmoc-Abz-OH), implement the step identical with preparation embodiment 1 and obtain title compound be
White powdery solids.
ESI-MS m/z：Calculated value 725.3, measured value 726.1 (M+H)+；HPLC purity assays, sample retention time tR=
14.153min (ratio of solvent acetonitrile and water increases to 90% in 30min from 10%, purity 100.00%)；Or tR=
20.321min (ratio of solvent methanol and water increases to 90% in 30min from 10%, purity 99.98%).
The test of Application Example biological activity
Experiment material and instrument：
Grb2-SH2 albumen is purchased from Santa Cruz companies of the U.S..Rink or PAL resins are purchased from Bachem companies of Switzerland.SA
Chip is purchased from Pharmacia Biacore companies of Sweden.
Embodiment 1Grb2-SH2 Binding experiments
The pharmacology test of molecular level of the present invention is using surface plasma resonance technology (Surface Plasmon
Resonance,SPR).Use Biacore3000 (Pharmacia Biosensor, Uppsala, Sweden) test ligand pair
The Competitive assays activity of Grb2-SH2 albumen.IC50Value is by testing the inhibitor of various concentration and the Grb2-SH2 albumen of recombination
Combination number when mixed solution and coupled SHC (pTyr-317) phosphoeptide effect on SA chips reach balance is calculated
(bibliography：Lyn,O.;Lung,F.-D.;Lakshmi,S.;James,B.;Roller,P.P.;King,C.R.Nonpho-
sphorylated peptide ligands for the Grb2 Src homology 2 domain.J.Bio
Chem.1997,272,29046–29052)。
2 cell experiment of embodiment
MDA-MB-453 cells are with (3-4) × 103The density of cell/plate is inoculated in 96 orifice plates, and cell divides completely after 4 days
It is melted into ripe.With WST-8 (2- (2- methoxyl group -4- nitre phenyl) -3- (4- nitre phenyl) -5- (2,4- disulfobenzenes) -2H- tetrazolium lists
Sodium salt) measure the inhibition of compounds on cell growth under various concentration.After 10% WST-8 (v/v) is added in every hole, after
Continue and cultivated 2-3 hours at 37 DEG C.Use absorbance of the TECANULTRA Reader test samples at 450nm.By comparing not
The absorbance of cell of cell (using DMSO as control) with being handled with compound of processing calculates half cytostatic concentration
(IC50)。
Compound activity obtained is shown in Table 1 and Fig. 1 in above-described embodiment：IC50Refer to half-inhibition concentration, i.e. enzymic catalytic reaction
Or the concentration of cell growth inhibitor when being suppressed half.
Table 1
As can be seen from the above table, the compound of the present invention 1-17 is effective inhibitor of Grb2-SH2, in 1- aminocyclohexyls
The nitrogen end connection hydrophobic amino acid fragment of base formic acid (Ac6c), as Leu, Phe or 4-Me-Phe are advantageous activity；Tetrapeptide
The protecting group of nitrogen end does not make significant difference to activity, and 11 compounds reach 10 μM to the inhibition of Grb2-SH2 in the series compound
It is horizontal.From the point of view of the test of MDA-MB-453 cell activity (table 1 and Fig. 1), compound 6 (LSG-105) and 12 (LSG-120) have
10 μM of horizontal activity.
Therefore, the compounds of this invention is effective Grb2-SH2 inhibitor, can be used as treating or preventing and Grb2-SH2 activation
Related disease, such as cancer, leukaemia or inflammation.
Claims (6)
1. have the following structure the open chain tetrapeptide analogs of the structure described in formula or its isomers or its is pharmaceutically acceptable
Salt：
2. prepared by open chain tetrapeptide analogs according to claim 1 or its isomers or its pharmaceutically acceptable salt
For treating or preventing the purposes in the drug of the disease as caused by the activation of Grb2-SH2.
3. purposes according to claim 2, wherein, disease caused by the activation as Grb2-SH2 is breast cancer, ovum
Nest cancer, brain tumor, prostate tumor, leukaemia, lymthoma, autoimmune disease, inflammation, metabolic disease or cardiovascular and cerebrovascular disease.
4. purposes according to claim 3, wherein, the metabolic disease is diabetes or obesity.
5. a kind of one or more open chain tetrapeptide analogs according to claim 1 or its isomery comprising therapeutically effective amount
The pharmaceutical composition of body or its pharmaceutically acceptable salt.
6. pharmaceutical composition according to claim 5 further includes acceptable excipient on galenic pharmacy.
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| CN1185785A (en) * | 1996-02-15 | 1998-06-24 | 法玛西雅厄普约翰公司 | Peptide antagonists of cellular mitogenesis and motogenesis and their therapeutic use |
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| CN1185785A (en) * | 1996-02-15 | 1998-06-24 | 法玛西雅厄普约翰公司 | Peptide antagonists of cellular mitogenesis and motogenesis and their therapeutic use |
Non-Patent Citations (2)
| Title |
|---|
| Discovery of a Novel Nonphosphorylated Pentapeptide Motif Displaying High Affinity for Grb2-SH2 Domain by the Utilization of 3′-Substituted Tyrosine Derivatives;YanLi Song等;《journal of medicinal chemistry》;20060207;第49卷;全文 * |
| 基于配体结构的Grb2-SH2抑制剂的结构优化：更高的活性、更少的电荷、更低的肽性;彭电等;《有机化学》;20110616;第31卷(第12期);全文 * |
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