CN106562946A - Benzoyl hydrazine allosteric inhibitor for D-3-phosphoglycerate dehydrogenase and applications of benzoyl hydrazine allosteric inhibitor - Google Patents
Benzoyl hydrazine allosteric inhibitor for D-3-phosphoglycerate dehydrogenase and applications of benzoyl hydrazine allosteric inhibitor Download PDFInfo
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- VZYMRJLQRNMRET-BJMVGYQFSA-N CN/N=C/c(cc(cc1)[N+]([O-])=O)c1O Chemical compound CN/N=C/c(cc(cc1)[N+]([O-])=O)c1O VZYMRJLQRNMRET-BJMVGYQFSA-N 0.000 description 1
- IIFCLXHRIYTHPV-UHFFFAOYSA-N COC(c(c(O)c1)ccc1O)=O Chemical compound COC(c(c(O)c1)ccc1O)=O IIFCLXHRIYTHPV-UHFFFAOYSA-N 0.000 description 1
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- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
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Abstract
The invention discloses a benzoyl hydrazine allosteric inhibitor for D-3-phosphoglycerate dehydrogenase and applications of the benzoyl hydrazine allosteric inhibitor, wherein the structure of the benzoyl hydrazine allosteric inhibitor is shown as the formula I, R1, R2, R3, R4, R5, R6 and R7 are the same or different, and respectively represent hydrogen, halogen, nitryl, hydroxyl, amino or substituted amino, alkyl, alkoxy, and benzyloxy or halogen substituted alkyl independently, or the adjacent substituent groups can form a ring. The in-vitro enzymatic activity testing, cell viability testing and mouse heterotransplantation model experiment prove that the compound can specifically inhibit the activity of D-3-phosphoglycerate dehydrogenase, and the growth of cancer cells is delayed by reducing the overexpression of D-3-phosphoglycerate dehydrogenase in the cancer cells. The compound can be used for treating, preventing or inhibiting the tumor diseases including breast cancer, colon cancer, melanoma and non-small cell lung cancer when used independently or in combination with other anti-cancer drugs. The formula I is shown in the description.
Description
Technical field
The present invention relates to treat and prevent due to the medicine of the disorderly caused various diseases of serine metabolism, more particularly to make
N ' for D-3- phosphoglycerate dehydrogenase inhibitor-substituted benzoyl hydrazide kind compound, and the compound and combinations thereof uses
Application of the medicine in treatment breast carcinoma, colon cancer, melanoma, nonsmall-cell lung cancer and Other diseases.
Background technology
D-3- phosphoglycerate dehydrogenases (PHGDH) the catalytic serine synthesis first step in human body, is serine synthesis
Key enzyme in path.PHGDH was proved the melanoma cell in the mankind 40% or 70% three negative breasts in 2011
There is the situation of overexpression in cancerous cell etc., the knockout experiment for carrying out PHGDH genes finds that these cancerous cell are in vivo and in vitro
Growth be largely suppressed【(1)Locasale,J.W.,Grassian,A.R.,Melman,T.,Lyssiotis,C.A.,
Mattaini,K.R.,Bass,A.J.,Heffron,G.,Metallo,C.M.,Muranen,T.,Sharfi,H.,et al.
(2011).Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes
to oncogenesis.Nat.Genet.43,869-874.(2)Possemato,R.,Marks,K.M.,Shaul,Y.D.,
Pacold,M.E.,Kim,D.,Birsoy,K.,Sethumadhavan,S.,Woo,H.K.,Jang,H.G.,Jha,A.K.,et
al.(2011).Functional genomics reveal that the serine synthesis pathway is
essential in breast cancer.Nature 476,346-350.】.Therefore, carried out using PHGDH as anticancer target
Drug design has bright prospects.Due to PHGDH active pocket small volumes, prothetic group NAD+In vivo at concentrations up to
0.3mM and PHGDH complete crystal structures such as do not solve so far at the reason, and the drug design based on PHGDH active pockets enters to postpone
Slowly.New thinking is to carry out the allosteric control of PHGDH, designs the inhibitor of PHGDH.
Allosteric control in protein can be described as other structure molecule causes albumen with reference to the nonactive site in albumen
The phenomenon that activity changes.The advantage of other structure medicine is which has high specificity, and regulation and control target proteinses are active rather than complete
Its loss of activity is made entirely, and which plays effect etc. in the presence of endogenous ligands.
There is document to point out：The combination such as the knockout of PHGDH genes and cancer therapy drug cisplatin, amycin can show in vivo and in vitro
Write improve cancer therapy drug biological activity [(3) Jing, Z., Heng, W., Xia, L., Ning, W., Yafei, Q., Yao, Z.,
and Shulan,Z.(2015)Downregulation of phosphoglycerate dehydrogenase inhibits
proliferation and enhances cisplatin sensitivity in cervical adenocarcinoma
cells by regulating Bcl-2and caspase-3,Cancer Biol.Ther.16,541-548.(4)Zhang,
X.,and Bai,W.(2016)Repression of phosphoglycerate dehydrogenase sensitizes
triple-negative breast cancer to doxorubicin,Cancer Chemother.Pharmacol.78,
655-659.), this is used there is provided reference with anti-cancer agent in combination to carry out PHGDH inhibitor.So far, have no PHGDH
Inhibitor enters the report of clinical research, also have no its with anti-cancer agent in combination using effect of drugs be reported.For PHGDH
Other structure site is carried out drug design and allosteric inbibitor is used for tumor prevention and treatment, with novelty and creativeness.
The content of the invention
It is an object of the invention to provide a kind of benzoyl hydrazine class compound, as the allosteric inbibitor of PHGDH.
The present invention also aims to providing above-mentioned benzoyl hydrazine class compound is treating and preventing some breast carcinoma, colon
Application in the diseases such as cancer, melanoma and nonsmall-cell lung cancer.
The present invention also aims to provide above-mentioned benzoyl hydrazine class compound with other PHGDH inhibitor or cancer therapy drug
Composite reagent is treated and prevented in the disease medicaments such as some breast carcinoma, colon cancer, melanoma and nonsmall-cell lung cancer in preparation
Application.
The present invention is by PHGDH protein surface property analysis, being found suitable for the potential other structure site (ginseng of small molecule combination
See Fig. 1), and virtual screening is carried out for institute's prediction bits point.It is different by vitro enzyme active testing, cytoactive test and mice
Transplantation model is planted, substituted benzoyl hydrazides molecule is proved to be PHGDH inhibitor.By co-precipitation and liquid chromatograph-efficient mass spectrum
Technology used in conjunction analytical control simultaneously demonstrates the specificity of this kind of compound.
What the present invention was provided can have following general structure as the compound of the allosteric inbibitor of PHGDH：
In Formulas I, R1、R2、R3、R4、R5、R6、R7It is identical or different, each independently represent hydrogen, halogen, nitro, hydroxyl, amino
Or substituted-amino, alkyl, alkoxyl, benzyloxy or halogen-substituted alkyl, or, wherein two adjacent substituent group (R1With R2、
R2With R3、R4With R5、R5With R6And/or R6With R7) can be with cyclization.
The halogen includes F, Cl, Br and I.
The substituted-amino preferably C1～C12 alkyl-substituted aminos, more preferably C1～C6 alkyl-substituted aminos, for example
Methylamino, ethylamino-, dimethylamino, diethylin etc..
The alkyl is preferably C1～C12 alkyl, more preferably C1～C6 alkyl, such as methyl, ethyl, propyl group, isopropyl
Base, butyl etc..
The alkoxyl is preferably C1～C8 alkoxyls, more preferably C1～C4 alkoxyls, such as methoxyl group, ethyoxyl,
Propoxyl group etc..C1～C12 alkyl of the halogen-substituted alkyl preferably one or more halogen substiuteds, more preferably one or many
C1～C6 alkyl of individual halogen substiuted, such as trifluoromethyl etc..
R1With R2、R2With R3、R4With R5、R5With R6And/or R6With R7During cyclization, Joint Representative's 1,3-butadiene-Isosorbide-5-Nitrae-two
Base, Isosorbide-5-Nitrae-dibutyl are jointly formed naphthalene, tetrahydronaphthalene etc. with the phenyl ring being located.
Compound of formula I can be prepared by the following method：
With substituted benzoyl hydrazides and substituted benzaldehyde dehydrating condensation, corresponding compound of formula I is obtained.
With compound (E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitrobenzals) benzoyl hydrazine (PKUMDL-WL-
2101), as a example by, its synthetic route is：
Other specific examples of compound of formula I can be found in the table 1 in embodiment 2.
Chemical substance used by this synthetic route is commercially available prod or can be obtained by prior art synthesis, reacted
When, the operational approach for being adopted and operating procedure and reaction condition and intermediate etc. are in accordance with those skilled in the art ripe
The methodology of organic synthesis design known, implement, and be disclosed in embodiment.
The present invention is tested by enzymatic activity, cell experiment and mice xenograft model are it is experimentally confirmed that chemical combination shown in Formulas I
Thing can specificity suppress PHGDH it is active.PHGDH is suppressed by the other structure of compound of formula I, it is possible to decrease mistakes of the PHGDH in cancerous cell
Expression, so as to delay the growth of cancerous cell.
By the substituted benzoyl hydrazide kind compound of the present invention individually, or with other PHGDH inhibitor or cancer therapy drug group
Medicine is shared, or using their pharmaceutical salts as effective ingredient, adds Conventional pharmaceutical carriers, can be prepared each for treating or preventing
Plant the medicine of cancer.
The pharmaceutical salts of the substituted benzoyl hydrazide kind compound of the present invention and combinations thereof medication refer to pharmaceutically acceptable salt,
The salt for for example being formed with the mineral acid such as hydrochloric acid, sulphuric acid, phosphoric acid, nitric acid, or with citric acid, succinic acid, citric acid, acetic acid, wine
The salt that the organic acid such as stone acid, methanesulfonic acid are formed.
Conventional pharmaceutical carriers refer to nontoxic solid-state, semisolid or liquid filler, diluent, adjuvant, lapping or other
Pharmaceutical adjunct.According to techniques known, can according to therapeutic purposes, route of administration need pharmaceutical composition is made
Various dosage forms.
Description of the drawings
Fig. 1 shows the other structure site of the PHGDH of protein surface property locator CAVITY prediction.
Fig. 2 is the molecular docking figure of compound PKUMDL-WL-2101 and PHGDH.
Fig. 3 shows the Cancer cell killing activity (A) of PKUMDL-WL-2101 in embodiment 4 and its breast cancer cell is had
The impact (B) of silk division cycle.
During Fig. 4 shows embodiment 5, biotin label does not interfere with inhibition of enzyme activity of the PKUMDL-WL-2101 to PHGDH
And PKUMDL-WL-2101 being capable of selectively targeted PHGDH (B) in cell (A).
Fig. 5 shows shadows of the PKUMDL-WL-2101 to serine network metabolite in breast cancer cell in embodiment 6
Ring, wherein A is metabolism network figure, B show compound PKUMDL-WL-2101 affect cell in serine and Glycine Metabolism produce
The amount of thing.
Fig. 6 shows in vivo bioactivities of the PKUMDL-WL-2101 in mice xenograft model in embodiment 7, its
In, A, B, C figure is administered 2 months tumor bodies when showing PKUMDL-WL-2101 dosages respectively 5,10,20mg/kg/day
Long-pending change；D shows the change of the Mouse Weight of different dosing dosage.
Fig. 7 shows PKUMDL-WL-2101 and furfuran compound PKUMDL-WL-2201 composite reagents in embodiment 8
Design sketch.
Specific embodiment
Following examples are used to illustrate the present invention that the method for the present invention to be put into practice in expression, and which is to the scope of the present invention without any
Limit.Those skilled in the art may find the obvious additive method for realizing the present invention for them, all should recognize
It is included in the scope of the present invention for those methods.
The discovery of embodiment 1, PHGDH allosteric inbibitors
First, the prediction in the other structure sites of PHGDH
PHGDH(PDB code:2G76) the prediction in other structure site, uses protein surface heuristic routine CAVITY.It is first
First, the program is detected to protein surface by wiping ball method, finds the potential binding site of protein surface；Subsequently, journey
Sequence rule of thumb formula (CavityScore=(Volume-AdjustVolume)/(SurfaceArea-
AdjustSurfaceArea)) ability of protein binding small molecule is given a mark.AdjustVolume and
AdjustSurfaceArea is related to the hydrophobic area of residue and hydrogen bond receptor donor quantity in predicted site.By to known
The maximum pK of binding site-ligand binding pairDGiven a mark, and with known experiment pKDValue is fitted, and obtains preferably linear
Dependency.Therefore, binding site-part maximum pK that program can will be given a mark to predict according to above-mentioned formulaDThe form of value is given.
According to pKDNumerical value and pocket volume size, it is final to choose suitable potential other structure site.For PHGDH, we are first
It is secondary to obtain two brand-new potential other structure sites：MDL-1 and MDL-2.As shown in figure 1, MDL-1 be located at avtive spot with
And prothetic group NAD+The vicinity of binding site, pocket volume isThe maximum pK for being predictedDFor 8.71.MDL-1 and active sites
Point shares 78 glycine, 79 L-Valine, 80 aspartic acids, 81 agedoites and 82 L-Valine.MDL-2 is located at
Substrate-binding domain, pocket volume size isThe maximum pK for being predictedDFor 7.79.The main research of the present invention is right
As for MDL-1.
2nd, the virtual screening of the other structure molecules of PHGDH
For the other structure site predicted, using the method for molecular docking, to the SPECS data comprising about 200,000 compounds
Storehouse carries out virtual screening.First, rough rigidity docking is carried out using Glide SP patterns, secondly, choose marking front 10,000
Compound, it is considered to compound and protein residues side chain flexibility, further docked using Glide XP patterns.Finally,
The compound for choosing Glide XP marking front 1000 carries out hand picking, and 49 compounds of purchase are used for experimental verification.These changes
Compound further verifies activity during enzymatic activity is tested in vitro.The activated compound that MDL-1 sites obtain is 5, IC50Value
Compound less than 50 μM is 1, is compound (E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitros of this research report
Benzal) benzoyl hydrazine, it is named as PKUMDL-WL-2101.The compound is shown in Fig. 2 with the result of docking of PHGDH.
The synthesis of embodiment 2, other structure molecule
First, the design of PKUMDL-WL-2101 analog
According to active ingredient compounds (E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitrobenzals) benzoyl hydrazine
(PKUMDL-WL-2101) the docking model combined with PHGDH (see Fig. 2), it is seen that the interaction mould of small molecule and PHDGH
Formula：Two phenyl ring occupy the hydrophobic cavity in pocket, and even on the phenyl ring of acyl group, the hydroxyls of 4 replacements can be with 261 Phenylalanine
Form hydrogen bond；The hydroxyl of 2 replacements can form hydrogen bond with 264 glutamic acid；Ketonic oxygen and 57 lysine shapes in hydrazides chain
Into hydrogen bond, hydrazides nitrogen can form hydrogen bond with 264 glutamic acid；On another phenyl ring, the nitro of 3 can be with 134 arginine or 55
Position alanine forms hydrogen bond；The ring also has electrostatic interaction with the cavity of positively charged around.Accordingly, we optimize two phenyl ring
Substituted radical, designs a series of PKUMDL-WL-2101 analog.
2nd, the synthesis of PKUMDL-WL-2101 and the like
With (E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitrobenzals) benzoyl hydrazine (PKUMDL-WL-2101) it is
Example, describes the synthesis of benzoyl hydrazine PHGDH inhibitor.
Synthetic route is：
Experimental procedure is：
(1) 2,4- methyl dihydroxy benzoates (1.781g, 11.5mmol) are dissolved in 50mL methanol solutions, thereto plus
Enter 85% hydrazine hydrate (2.031g, 34.5mmol), back flow reaction, TLC detection reactions disappear to raw material.Cooling, vacuum distillation
Solvent is removed, residue is recrystallized to give target product 2 in methyl alcohol, and (white solid is produced 4- dihydroxybenzoyl hydrazine 1.546g
Rate 80%),1H-NMR(400MHz,DMSO):6.35 (2H, m), 7.77 (1H, d, J=9.20Hz), 10.06 (1H, s), 10.69
(1H,s),11.86(1H,s)。
(2) by (1) products therefrom 2,4- dihydroxybenzoyl hydrazines (1.681g, 10.0mmol) and 5- nitrosalicylaldehydes
(1.670g, 10.0mmol) is stirred in 50mL methanol solutions, and normal-temperature reaction disappears to TLC detection reactions to raw material.Decompression is steamed
Solvent is evaporated, target product (E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitro benzal in residue methanol, is recrystallized to give
Base) benzoyl hydrazine 2.378g (yellow solid, yield：75%, fusing point：296-298 DEG C),1H-NMR(DMSO):6.33(1H,d,J
=1.80Hz), 6.39 (1H, dd, J=1.98,8.92Hz), 7.12 (1H, d, J=8.99Hz), 7.81 (1H, d, J=
8.75Hz), 8.18 (1H, dd, J=2.64,9.20Hz), 8.57 (1H, d, J=2.57Hz), 8.73 (1H, s), 10.26 (1H,
s),12.02(1H,s),12.15(1H,s),12.32(1H,s)；13C NMR(101MHz,DMSO-d6)δ165.32,162.95,
162.55,162.19,144.51,139.85,129.84,126.47,123.83,119.78,117.06,107.58,105.83,
102.81.Mass spectrum：ESI-MS measured values (theoretical value [M+H+]) 318.1 (318.1).
31 benzoyl hydrazine class compounds are prepared using said method, characterize data lists table 1 in.Nmr spectrum data
(1H-NMR) measured by U.S. Varian Mercury 400M.Using sub- for the deuterated dimethyl of interior target containing tetramethylsilane
Used as solvent, using Hz as unit, abbreviation used is illustrated as coupling constant sulfone：S=is unimodal, and d=is bimodal, t=triplets, q=
Quartet, m=multiplets, the mono- broad peaks of br=.Fusing point is determined by Beijing Tyke Instrument Ltd. X-4 numerical monitors micro melting point
Instrument is measured.
1. noval chemical compound of table is characterized
Respectively numbering corresponding compound name is：
1)PKUMDLWL-2101：(E) -2,4- dihydroxy-N'- (2- hydroxyl -5- nitrobenzals) benzoyl hydrazine；
2)PKUMDLWL-2102：(E)-N'- (4- fluorine benzals) benzoyl hydrazine；
3)PKUMDLWL-2103：(E)-N'- benzals -2,4- dihydroxybenzoyl hydrazines；
4)PKUMDLWL-2104：(E) -2,4- dihydroxy-N'- (naphthalene -1- methylene) benzoyl hydrazine；
5)PKUMDLWL-2105：(E) -2,4- dihydroxy-N'- (4- nitrobenzals) benzoyl hydrazine；
6)PKUMDLWL-2106：(E)-N'- (2- hydroxyl -5- nitrobenzals) -4- nitrobenzoyl hydrazides；
7)PKUMDLWL-2107：(E)-N'- (2- hydroxyl -5- nitrobenzals) -1- naphthols hydrazides；
8)PKUMDLWL-2108：(E)-N'- (2- hydroxyl -5- nitrobenzals) -4- toluyl hydrazines
9)PKUMDLWL-2109：(E) -2,4- dihydroxy-N'- (4- hydroxyl benzals) benzoyl hydrazine
10)PKUMDLWL-2110：(E) -2- hydroxy-ns '-(2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine；
11)PKUMDLWL-2111：(E) the fluoro- N'- of -4- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
12)PKUMDLWL-2112：(E) -2,4- dihydroxy-N'- (3- benzylidenes) benzoyl hydrazine
13)PKUMDLWL-2113：(E)-N'- (4- ethyoxyl -3- nitrobenzals) -2,4- dihydroxybenzoyl hydrazines
14)PKUMDLWL-2114：(E) -2,4- dihydroxy-N'- (3- nitrobenzals) benzoyl hydrazine
15)PKUMDLWL-2115：(E) -3- hydroxy-ns '-(2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
16)PKUMDLWL-2116：(E) -2,4- dihydroxy-N'- (4- methoxyl group -3- nitrobenzyls) benzoyl hydrazine
17)PKUMDLWL-2117：(E) -2,4- dihydroxy-N'- (3- hydroxyl benzals) benzoyl hydrazine
18)PKUMDLWL-2118：(E) -4- hydroxy-ns '-(2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
19)PKUMDLWL-2119：(E) the chloro- N'- of -3- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
20)PKUMDLWL-2120：(E)-N'- (2- hydroxyl -5- nitrobenzals) -3- nitrobenzoyl hydrazides
21)PKUMDLWL-2121：(E) -4- amino-N'- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
22)PKUMDLWL-2122：(E)-N'- (2- hydroxyl -5- nitrobenzyls) -2- toluyl hydrazines
23)PKUMDLWL-2123：(E) -4- methoxyl groups-N'- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
24)PKUMDLWL-2124：(E) -4- (tert-butyl group)-N'- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
25)PKUMDLWL-2125：(E) the bromo- N'- of -4- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
26)PKUMDLWL-2126：(E)-N'- (2- hydroxyl -5- nitrobenzals) -3- methoxybenzoyl hydrazines
27)PKUMDLWL-2127：(E) the chloro- N'- of -4- (2- hydroxyl -5- nitrobenzyls) benzoyl hydrazine
28)PKUMDLWL-2128：(E)-N'- (2- hydroxyl -5- nitrobenzyls) -4- (trifluoromethyl) benzoyl hydrazine
29)PKUMDLWL-2129：(E)-N'- (4- chlorine benzals) -2,4- dihydroxybenzoyl hydrazines
30)PKUMDLWL-2130：(E)-N'- (4- chlorine benzals) -2,4- dihydroxybenzoyl hydrazines
31)PKUMDLWL-2131：E)-N'- (4- bromine benzals) -2,4- dihydroxybenzoyl hydrazines
32)PKUMDLWL-2132：(E) -2,4- dihydroxy-N'- (2- nitrobenzals) benzoyl hydrazine
Embodiment 3, fluorescence kinetics method determine the external enzymatic activitys of PHGDH of PKUMDL-WL-2101 and the like
The measure of PHGDH enzymatic activitys is realized by detecting fluorescence emission spectrums of the NADH at 456nm.First,
PHGDH (final concentration 30ng/ μ L) in 96 orifice plates with HEPES buffer solution (25mM, pH 7.1,400mM KCl), 5 μM of PLP,
0.5mM α KG, 150 μM of NADH and PSAT1 (final concentration 30ng/ μ L) are incubated 10 minutes.Subsequently, 10 μ L DMSO (controls are added
Group) or the DMSO solution containing small molecule, balance is shaken 5 minutes with the rotating speed of 550rpm at 25 DEG C.Enzyme activity body test system
The final concentration (v/v) of middle holding DMSO is 5%.Finally, add Pser aqueous solutions (final concentration 0.5mM), start reaction, and with purple
Outer visible microplate reader monitors the consumption of NADH under 456nm over time.Albumen is lived using first rate is reacted within 30s
Property be estimated, now NADH is consumed and is increased linear with the time.
Table 3.PKUMDL-WL-2101 and the like vitro enzyme active testing results
The dose-effect relationship of further investigation compound, obtains part IC50Numerical value, the IC of PKUMDL-WL-210150For 34.8 ±
3.6 (μM), the IC of PKUMDL-WL-212850For 36.1 ± 4.2 (μM).
The cancer cell suppression activity of embodiment 4, compound
From a series of cancerous cell and normal mammary epithelial, using MTT (3- (and 4,5)-
Dimethylthiahiazo (- z-y1) -3,5-di-phenytetrazoliumromide) experimental technique, compound is existed
Biological activity on cellular level is studied.Specific practice is：First, by the PHGDH sensitivities in exponential phase growth
Breast cancer cell MDA-MB-468 (5000 cells/well) and HCC70 (5000 cells/well), PHGDH insensitive breast cancer cell
MCF-7 (3000 cells/well), MDA-MB-231 (2000 cells/well), ZR-75-1 (4000 cells/well), colon cancer cell
DLD-1 (2000 cells/well) and normal mammary epithelial MCF-10A (3000 cells/well) are transferred in 96 orifice plates, are overnight pasted
Wall.Then, add the compound of variable concentrations into 96 orifice plates, and cell incubation 72 hours, control DMSO final concentrations (v/v) is
0.2%.DMSO without any compound is used as control.Subsequently, after 72 hours, 20 μ L 5mg/mL are added in each experimental port
MTT, after being incubated at least 4 hours, removes the liquid in each experimental port, adds 200 μ L DMSO, and 37 DEG C are slowly rocked 10 minutes,
Using microplate reader detect 490nm at can be with light absorbs.Experimental data represented using cell survival rate %, cell median lethal
Rate EC50Value is obtained by Hill equation models.
PKUMDL-WL-2101 shows micromolar inhibitory activity on a cellular level (see A in Fig. 3).Which is to PHGDH
The EC that sensitive breast cancer cell MDA-MB-468 and HCC70 shows50Value is respectively 7.70 and 10.8 μM, to PHGDH
Insensitive breast cancer cell MDA-MB-231, the EC that ZR-75-1 and MCF-7 show50Value be respectively 27.8,83.4 and
139 μM, the EC shown by colon cancer cell50It is worth for 18.3 μM.Meanwhile, PKUMDL-WL-2101 shows faint cell toxicant
Property, the EC gone out by MCF-10A cells shows50It is worth for 45.8 μM.
By the MDA-MB-468 cells (3 × 10 by the exponential growth cycle is in5Cells/well) it is transferred in 6 orifice plates, mistake
The night adherent compound incubation for adding variable concentrations after 24 hours, fix by pancreatin digestion, centrifugation, the ethanol of 70% pre-cooling, PBS punchings
Wash, be centrifuged, resuspended (PBS of 0.5%triton-x-100,50 μ g/ml PI and 50 μ g/ml DNase-free RNase),
37 DEG C of lucifuges 30 minutes, show using flow type analyzer analysis result, PKUMDL-WL-2101 makes cell cycle arrest in G0/G1
Phase (see B in Fig. 3).
Above test result indicate that, PKUMDL-WL-2101 sensitive to PHGDH breast cancer cell shows preferably thin
Born of the same parents' killing activity.
Embodiment 5, PKUMDL-WL-2101 targeting PHGDH in cell
First, MDA-MB-468 lysates biotin labeling are fixed on M-280 strepavidin magnetic beads with carrying
PKUMDL-WL-2101 is incubated at room temperature 1 hour.Then, using 200 μ LPBST, (20) PBS buffer, 0.5%Tween delay
Three flushing magnetic beads of liquid are rushed, supernatant is abandoned in centrifugation.Finally, after using the resuspended M-280 strepavidin magnetic beads of PBST buffer, take appropriate muddy
Turbid liquid is mixed with 5 × SDS buffer, 80 DEG C, and centrifuging and taking supernatant after 10min carries out PHGDH inspections using WB technologies.Simultaneously will
MDA-MB-468 lysates, M-280 strepavidin magnetic beads after the incubation of MDA-MB-468 lysates and biotin and not any
Used as control, wherein lysate directly carries out PHGDH inspections using WB technologies for the M-280 strepavidin magnetic beads incubation of fixing process,
The process of the magnetic bead element of test organisms first PHGDH enzymatic activity of the label to PKUMDL-WL-2101 identical with sample sets in matched group
Suppression ratio does not affect (see A in Fig. 4), and the structure of the PKUMDL-WL-2101 with biotin label is as follows, meanwhile, phase
Compared with matched group, only there is co-precipitation with the PKUMDL-WL-2101 with biotin label, also indicate that, PKUMDL- in PHGDH
(see in Fig. 4 B) that really with PHGDH combined of the WL-2101 in cell, wherein passage 1 and 2 are cell pyrolysis liquid loading, are led to
Road 3 and 4 is that the PKUMDL-WL-2101 for being fixed on M-280 strepavidin magnetic beads surface is incubated with cell pyrolysis liquid, washing, after boiling
Supernatant loading, passage 5 and 6 is to be fixed on the biotin on M-280 strepavidin magnetic beads surface to be incubated with cell pyrolysis liquid, washing,
Supernatant loading after boiling, passage 7 and 8 are that the M-280 strepavidin magnetic beads for not making any fixing process are incubated with cell pyrolysis liquid,
Washing, the supernatant loading after boiling.Above test result indicate that, compound PKUMDL-WL-2101 targeting PHGDH in cell.
Impact of the embodiment 6, PKUMDL-WL-2101 to metabolite in serine metabolism network
First, by MDA-MB-468 cells (3 × 105Cells/well) plant in 6 orifice plates, Jing after 24h is adherent, by cell
In culture medium be replaced as adding dialysis serum and 11mMU-13The fresh culture of C- glucose markers, while adding 5 μ Μ
Continue culture 24h after PKUMDL-WL-2101.Subsequently, culture fluid is removed, 6 orifice plates is cleaned using 1 × PBS solution, add 1mL-
80 DEG C of 80% methanol aqueous solutions for pre-cooling, after -80 DEG C of 15min, cell are scraped from 6 orifice plates, centrifuging and taking supernatant.Most
Afterwards, the supernatant in each hole is dried up under Nitrogen evaporator, solid 15 μ L methanol/acetonitrile (1:It is 1v/v) resuspended, pass through liquid phase after centrifugation
Color-High Resolution Spectrum mass spectrum connection (LC-HRMS) detection Metabolites Concentration (see A in Fig. 5).
By experimental result as can be seen that add compound after, the conjunction of serine and glycine in MDA-MB-468 cells
Into being suppressed, decrement is about 50% (see B in Fig. 5).
Above experimental result again shows that, compound PKUMDL-WL-2101 targeting PHGDH and notable shadow in cell
Ring the amount of serine and Glycine Metabolism product.
Biological activity effect of the embodiment 7, PKUMDL-WL-2101 in mice xenograft model
First, by MDA-MB-468 (2 × 105) cell infusion is to NOD.CB17-PrkdcscidThe of/J mices (6-8 is all)
In four mammary fat pads, treat tumor average volume length to 30mm3, mice is randomly divided into into 8 groups, per group of 5 mices.Subsequently, open
Begin to be administered, solvent (10%DMSO, 20%EL and 70%PBS) of the matched group administration for dissolved compound, PKUMDL-WL-
2201 administering mode be gavage, dosage be respectively 20,10 and 5mg/kg/day.Detect that per two days gross tumor volume is big
Little, gross tumor volume passes through equation below：
Minor axis2(mm) × major diameter (mm) × 0.5
It is calculated.
PKUMDL-WL-2101 is administered two months, and experimental result is as shown in Figure 6, it can be seen that compared to matched group, mice
Internal tumour growth is suppressed significantly (see A-C in Fig. 6).In experimentation, mice keeps preferable growth conditions (see Fig. 6
Middle D).Experimental result has statistical significance, and P values are less than 0.05.
The composite reagent of embodiment 8, PKUMDL-WL-2101 and furfuran compound PKUMDL-WL-2201
The chemical name of furfuran compound PKUMDL-WL-2201 is：(E) the chloro- 4- of -2- (5- ((2- (ethylamino first
Acyl group) hydrazono-) methyl) furan -2- bases) benzoic acid, structural formula is as follows：
The specific practice of experiment is as follows：In enzyme activity assay experiment, the Concentraton gradient of PKUMDL-WL-2101 is
0,1,5,12.5,25,50,100,200 μM, choose successively one of concentration and PKUMDL-WL-2201 variable concentrations (0,
1,5,12.5,25,50,100,200 μM) combination of two, then it is incubated with PHGDH, detects shadow of the mixture to PHGDH enzymatic activitys
Ring.In cell activation assay experiment, first, MDA-MB-468 (5000 cells/well) is transferred to overnight adherent in 96 orifice plates；Its
It is secondary, choose successively a concentration in PKUMDL-WL-2101 variable concentrations (0,0.1,0.5,1,2.5,5,7.5,10 μM) with
The variable concentrations (0,0.1,0.5,1,2.5,5,7.5,10 μM) of PKUMDL-WL-2201 mix two-by-two；Finally, mixture is added
With cell incubation 72 hours in 96 orifice plates, MTT methods detection mixing microbic activity.
Enzyme activity assay test result indicate that, when the concentration of PKUMDL-WL-2201 is when 50 μM increase to 200 μM, collaboration
Phenomenon is substantially observed that it is 5 μM that the point (CI=0.34) for cooperateing with degree most strong is occurred in when the concentration of PKUMDL-WL-2101,
And the concentration of PKUMDL-WL-2201 be 200 μM when (see A in Fig. 7).From unlike enzymatic activity experimental result, in cytoactive
In test, then significantly there is (see B in Fig. 7) at PKUMDL-WL-2101 concentration maximas under experimental conditions in cooperative phenomenon.Enzyme
The reason for activity experiment result is different from cytoactive test result are likely due to what the microenvironment of intracellular complexity was caused.
Comprehensive enzymatic activity test, cell experiment and mice xenograft model experimental result, the compound of the present invention can be special
The opposite sex suppresses PHGDH active.
Claims (8)
1. the purposes of compound shown in Formulas I and its pharmaceutical salts in preparing for the medicine treating, prevent or suppress tumor：
Wherein, R1、R2、R3、R4、R5、R6、R7It is identical or different, each independently represent hydrogen, halogen, nitro, hydroxyl, amino or replacement
Amino, alkyl, alkoxyl, benzyloxy or halogen-substituted alkyl, or, R therein1With R2、R2With R3、R4With R5、R5With R6, and/
Or R6With R7Cyclization.
2. purposes as claimed in claim 1, it is characterised in that the substituted-amino is C1～C12 alkyl-substituted aminos.
3. purposes as claimed in claim 1, it is characterised in that the alkyl is C1～C12 alkyl；The alkoxyl be C1～
C8 alkoxyls.
4. purposes as claimed in claim 1, it is characterised in that the halogen-substituted alkyl is one or more halogen substiuteds
C1～C12 alkyl.
5. purposes as claimed in claim 1, it is characterised in that the R1With R2、R2With R3、R4With R5、R5With R6, and/or R6With
R7Cyclization refers to that two adjacent substituent groups connect into 1,3-butadiene-Isosorbide-5-Nitrae-diyl or Isosorbide-5-Nitrae-dibutyl.
6. purposes as claimed in claim 1, it is characterised in that compound shown in the Formulas I is following compounds PKUMDL-
One of WL-2101 to PKUMDL-WL-2132：
7. purposes as claimed in claim 1, it is characterised in that the tumor is breast carcinoma, colon cancer, melanoma or non-little
Cell lung cancer.
8. in claim 1 compound shown in Formulas I and its pharmaceutical salts in the inhibitor for preparing D-3- phosphoglycerate dehydrogenases
Application.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610926351.4A CN106562946A (en) | 2016-10-31 | 2016-10-31 | Benzoyl hydrazine allosteric inhibitor for D-3-phosphoglycerate dehydrogenase and applications of benzoyl hydrazine allosteric inhibitor |
| US16/344,799 US20200054593A1 (en) | 2016-10-31 | 2016-12-30 | D-3-phosphoglycerate dehydrogenase allosteric inhibitor and use thereof |
| PCT/CN2016/113476 WO2018076537A1 (en) | 2016-10-31 | 2016-12-30 | D-3-phosphoglycerate dehydrogenase allosteric inhibitor and use thereof |
| US16/405,569 US10722489B2 (en) | 2016-10-31 | 2019-05-07 | D-3-phosphoglycerate dehydrogenase allosteric inhibitor and use thereof |
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| CN201610926351.4A CN106562946A (en) | 2016-10-31 | 2016-10-31 | Benzoyl hydrazine allosteric inhibitor for D-3-phosphoglycerate dehydrogenase and applications of benzoyl hydrazine allosteric inhibitor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107827776A (en) * | 2017-11-10 | 2018-03-23 | 上海应用技术大学 | Acylhydrazone, preparation method and applications with antitumor activity |
| CN110759833A (en) * | 2019-10-25 | 2020-02-07 | 国家林业和草原局竹子研究开发中心 | Preparation method and application of 4-hydroxy-benzyl (2' -hydroxy-benzylidene) -hydrazide |
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| US20100035932A1 (en) * | 2008-08-07 | 2010-02-11 | Schepetkin Igor A | Novel formyl peptide receptor like 1 agonists that induce macrophage tumor necrosis factor alpha and computational structure-activity relationship analysis of thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20100035932A1 (en) * | 2008-08-07 | 2010-02-11 | Schepetkin Igor A | Novel formyl peptide receptor like 1 agonists that induce macrophage tumor necrosis factor alpha and computational structure-activity relationship analysis of thereof |
Non-Patent Citations (2)
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| JASON W LOCASALE1等: "Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis", 《NATURE GENETICS》 * |
| RICHARD POSSEMATO等: "Functional genomics reveal that the serine synthesis pathway is essential in breast cancer", 《NATURE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107827776A (en) * | 2017-11-10 | 2018-03-23 | 上海应用技术大学 | Acylhydrazone, preparation method and applications with antitumor activity |
| CN110759833A (en) * | 2019-10-25 | 2020-02-07 | 国家林业和草原局竹子研究开发中心 | Preparation method and application of 4-hydroxy-benzyl (2' -hydroxy-benzylidene) -hydrazide |
| CN110759833B (en) * | 2019-10-25 | 2022-06-14 | 国家林业和草原局竹子研究开发中心 | Preparation method and application of 4-hydroxy-benzyl (2' -hydroxy-benzylidene) -hydrazide |
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