CN104744375A - Guanidine NTR1 micromolecular antagonist - Google Patents

Abstract

A guanidine NTR1 small molecule antagonist. The invention relates to the fields of drug design and pharmacology. The present invention provides a compound with the following general formula I through virtual screening and cell activity testing and receptor-based structural modification, wherein, R ₁ is a hydrophobic group; R ₂ is a substituted aromatic ring; R ₃ is a polar substituent group. The compound has inhibitory activity on NTR1, and has an important application in preparing medicines for diseases related to NTR1 protein.

Description

A kind of guanidine NTR1 small molecule antagonist

technical field

The present invention relates to the fields of drug design and pharmacology, in particular to the structure of a series of guanidine NTR1 small molecule inhibitors obtained through virtual screening, cell activity testing, and receptor-based structural modification.

Background technique

G Protein-Coupled Receptor (GPCR) has seven transmembrane structures, is widely distributed in the body, and participates in the response to various signaling molecules. GPCR and its signal transduction system are usually in a dynamic balance, and according to the changes of various physiological factors, the regulation of downstream signaling pathways is initiated to maintain the stability of the intracellular environment; under pathological conditions, cells also receive external signals through GPCR to affect cell life. Activities are adjusted. Especially in malignant tumor cells, GPCR regulates the occurrence, growth, invasion and metastasis of tumor cells by mediating the signal transduction process of tumor cells. In almost all malignant tumor cells, certain types of tumor cells can be detected. Abnormal expression of GPCR molecules. Excessive activation or inhibition of GPCRs may lead to the disorder of intracellular signal transduction pathways, resulting in disordered physiological responses and abnormal physiological phenomena.

Neurotensin receptor 1 (NTR1), a member of class A in the GPCR superfamily, is mainly distributed in the central nervous system and gastrointestinal system, mediates the intracellular Gq protein signal transduction pathway, and regulates the concentration of intracellular calcium ions. The protein's natural agonist, neurotensin (NT), is a 13-peptide in which six amino acids Arg-Arg-Pro-Tyr-Ile-Leu play key roles. In recent years, it has been found that NT and NTR1 play key roles in the growth, proliferation, invasion and metastasis of various malignant tumors (breast cancer, pancreatic cancer, colon cancer, lung cancer, prostate cancer, etc.). It has been reported in the literature that NT is expressed in both normal breast epithelial cells and invasive breast cancer tissues, while NTR1 is only highly expressed in breast cancer tissues and tumor cell lines, and the expression rate of NTR1 in invasive ductal carcinoma of the breast is as high as 91%. Animal experiments have confirmed that NTR1 can promote the growth of transplanted tumor cells and shorten the doubling time of tumor cells, and induce the invasion and metastasis of transplanted tumors. Therefore, NTR1 antagonists can inhibit the occurrence and development of tumors to a certain extent, and are expected to become effective drugs for treating tumors and other diseases. The crystal structure of mouse NTR1 was successfully resolved in 2012, which provides favorable conditions for receptor-based drug design and compound optimization.

Our laboratory has previously established a database containing 10,000 small molecules targeting NTR1, which effectively increased the screening speed. At present, a number of antagonists targeting NTR1 targets have been reported in the literature. Starting from known antagonists, it is helpful to establish pharmacophore models and 2D-Fingerprint screening methods to find new small molecule antagonists.

Contents of the invention

The existing research and development of new drugs targeting NTR1 has not yet produced marketed drugs, and since the crystal structure of NTR1 has not been resolved at that time, drug screening and design can only be carried out based on the three-dimensional structure obtained by homology modeling. The purpose of the present invention is to perform virtual screening on the NTR1 small molecule database obtained by integrating multiple compound databases based on the analyzed three-dimensional crystal structure of the mouse NTR1 protein, and to test the cell activity of the screening results, and to conduct a cell activity test on the small molecules with activity based on Structural modification of receptors to find small molecular compounds with inhibitory activity on NTR1, which are used to prepare drugs for diseases related to NTR1.

The present invention obtains a guanidine compound through virtual screening, cell activity testing and structural modification, the structure of which is shown in general formula I.

in:

R ₁ is morpholine ring, unsubstituted and methyl, halogen, hydroxyl substituted C ₅ -C ₆ cycloalkane group, piperidine ring, benzene ring, pyridine ring, dimethylamino, isopropyl, tert-butyl hydrophobic Sexual group; R ₂ is a benzene ring or pyridine ring-like aromatic ring substituted by halogen, methyl or ethyl; R ₃ is hydrogen, or an amino, hydroxyl, aminomethyl or hydroxymethyl polar group; n for 1-3.

The guanidine compound provided by the invention can be applied to the preparation of medicines for diseases related to NTR1 protein.

The small molecular compound 1 with inhibitory activity screened by the present invention and obtained through the cell activity test is R ₁ is a morpholine ring, R ₂ is a para-chloro substituted benzene ring, R ₃ is hydrogen, n is 2, and its structure is as follows (compound 1), the IC ₅₀ value is 44.49 μM.

Advantages and beneficial effects of the present invention:

In the present invention, through virtual screening and cell activity testing, the results prove that the NTR1 small molecule antagonist (compound 1) obtained through the screening in the present invention has better inhibitory activity on intracellular calcium ion concentration. Furthermore, by analyzing the hydrophilic and hydrophobic properties, hydrogen bond donors and acceptors, charge properties, etc. of the pocket where NTR1 binds to the ligand, according to the docking results and scoring of compound 1 and NTR1, group additions, deletions and substitutions were carried out at certain positions, and Docking verification was carried out to carry out structural modification and modification of compound 1 to obtain a series of compounds, which can participate in its regulatory mechanism by acting on NTR1 in the body, and are used for the preparation or adjuvant treatment of tumors, Parkinson's disease, and schizophrenia and other diseases, and has good prospects for drug application.

Description of drawings

Figure 1 is a schematic diagram of the interaction between Compound 1 and NTR1.

Figure 2 is a schematic diagram of the interaction between compound 2 and NTR1 after modification

Figure 3 is a graph of Ca2 ⁺ fluorescence values for compound 1 versus logarithm of compound concentration.

Detailed ways

Various aspects and features of the present invention are further described below. But these examples are not meant to limit the present invention in any way.

Example 1, Virtual Screening and Cell Viability Testing of NTR1 Small Molecule Inhibitors

One, the method for virtual screening of the present invention comprises following 4 steps:

Step 1: Construction of NTR1 Small Molecule Database

Integrate multiple commercial small molecule databases (including ZINC, SPECS, J&K Screening Library, InterBioScreening, Timtec, Msdiscove, Iris-biotech, TCM Database, Ambinter Natural Products, AnalytiCon Discovery NP, Princeton NP, Molecular Diversity Preservation International, PUBCHEM), using Pipeline pilot The software removes repetitive molecules and molecules with similar structures, and selects compounds with good absorption and low toxicity according to the calculated ADMET properties of the molecules for Clustering, obtaining a database of 1 million small molecules. The Glide module in Schrodinger was used to dock these small molecules with NTR1 with SP precision, and the top 10,000 small molecules scored in the results were selected to form the NTR1 small molecule database.

Step 2: Construction and screening of pharmacophore model

Find 6 compounds with good activity and large structural differences from the BindingDB database (as shown below) as the training set, use the Create Pharmacophore Automatically module in Discovery Studio 3.5 to establish a pharmacophore model, and verify the test set and FitValue The values determined the most suitable pharmacophore model, which included 2 hydrophobic centers, 1 aromatic ring center, and 1 hydrogen bond donor. The NTR1 small molecule database was screened using Search 3D Database.

Step 3: Fingerprint Screening

The 3 active compounds (structural formulas are as follows) and 11 inactive compounds obtained in BindingDB were respectively used in the Fingerprint module in Discovery Studio3.5 to perform 2D-Fingerprint with the NTR1 small molecule database, and the structures and inactive compounds in the Fingerprint results of the active compounds were compared Compounds with similar structures were removed from the compound Fingerprint results, and compounds with unpublished and unreported structures were selected from them.

Step 4: Cluster Analysis

The results of the pharmacophore model screening in step 2 and the Fingerprint screening in step 3 were clustered and analyzed using the Cluster module under the Design and Analyze Libraries in DiscoveryStudio3.5, and were divided into 99 categories, and 1 was selected from each category. molecules, 99 compounds were obtained. These compounds were purchased and tested for cell viability.

Two, the cell activity test method of the present invention comprises following 2 steps:

Step 1: Construction of NTR1 stably transfected CHO cell expression system

CHO cells were cultured with RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO ₂ , and saturated humidity. Transform, amplify, and extract the recombinant plasmid PUC57-NTR1 from the DH5α glycerolbacteria containing the PUC57-NTR1 recombinant plasmid (enzyme cutting sites are EcoR Ⅰ and Hind Ⅲ), and double-digest PUC57-NTR1 and the expression vector pcDNA3.1(+) , recover the target gene NTR1 and the expression vector pcDNA3.1(+), connect NTR1 and pcDNA3.1(+), transform the ligation product into DH5α competent bacteria, select positive clones with ampicillin, amplify, extract the plasmid, and name it pcDNA3.1(+)-NTR1. CHO cells were transfected with pcDNA3.1(+)-NTR1 recombinant plasmid, and CHO cells transfected only with pcDNA3.1(+) expression vector were used as positive control. The stably transfected CHO cells were screened with G418 for cloning, subcultured and expanded to obtain a monoclonal human line.

Step 2: Test the activity of the compound on NTR1 stably transfected CHO cells

NT is the natural ligand of NTR1. After NT is combined with NTR1, the signal transduction pathway activated by the coupled Gq protein can cause an increase in the intracellular calcium ion concentration, and the small molecule compound with inhibitory activity on NTR1 can inhibit the cellular Therefore, whether the small molecule compound has inhibitory activity on NTR1 can be judged by detecting whether the intracellular calcium ion concentration increases after adding NT and the small molecule compound. SR48692 is a reported selective antagonist of NTR1, which can antagonize the activity of NT.

Using the calcium 6 kit, a preliminary experiment was performed first. After adding 1nM NT in the 96-well plate, add 10μM and 100μM test compounds respectively, measure the calcium flux value of each compound at 10μM and 100μM concentration, select the compound with inhibitory activity at 10μM, and preliminarily estimate its approximate IC ₅₀ value. Then take this IC ₅₀ value as the center, draw 8 concentration gradients at a ratio of 1:2, measure the calcium flux value of the compound at different concentrations, and use NT and SR48692 as negative and positive controls respectively to obtain the calcium ion fluorescence value - Compound concentration profile. According to the curve, GraphPad Prismversion 5.0 software was used to calculate the IC ₅₀ value of small molecule compounds.

Using the above-mentioned virtual screening and cell viability assay methods, a small molecule compound (compound 1) with inhibitory activity on NTR1 was screened and obtained: 1-(4-chlorophenoxy)-3-{2-imine-3-[ 2-(4-morpholine)ethyl]-2,3-dihydro-1-benzimidazolyl}-2-propanol with an IC ₅₀ value of 44.49 μM ( FIG. 3 ).

Example 2. Structural modification based on the mode of action between the small molecule and the receptor NTR1.

Firstly, open the three-dimensional crystal structure of NTR1 protein in Discovery Studio3.5, the structure comes from PDB (Protein Data Bank), and the PDB number is 4GRV. Hydrogenate it, remove water, delete the inserted T4 lysozyme structure, use the Prepare Protein module to prepare the protein, and use Define Site to find the active site according to the NT in the ligand. Small molecules were prepared using the Prepare Ligands module. After the preparation is completed, the Libdock method of the Dock Ligands module is used for docking, and the docking result is shown in Figure 1. According to the docking results, compound 1 forms hydrogen bonds with the key amino acids Arg327 and Tyr146 of the receptor reported in the literature, and the bond lengths of the hydrogen bonds are (FIG. 1), this hydrogen bonding is very important for the combination of the two. The chlorine on the chlorine-substituted benzene ring is replaced by isopropyl (compound 2), and the key hydrogen bond lengths of these two places are shortened to (figure 2). The shorter the bond length, the stronger the hydrogen bond, and the tighter the binding between the ligand and the receptor, the better the activity of the ligand may be. The activity of the modified compound 2 was measured by the cell activity test method in Example 1, and its IC ₅₀ value was calculated. The measured IC ₅₀ value of compound 2 was 39.62 μM.

Here, this modification is taken as an example to illustrate a specific implementation method, but not as a limitation.

Claims (7)

1. a guanidine class NTR1 small molecular antagonists, has such as formula the structure shown in I,

Wherein, R ₁it is hydrophobic group; R ₂it is substituted aroma ring; R ₃hydrogen or polar substitution group.

2. the guanidine class NTR1 small molecular antagonists according to claims 1, is characterized in that, described hydrophobic group is morpholine ring, do not replace and C that methyl, halogen, hydroxyl replace ₅-C ₆cycloalkyl group, piperidine ring, phenyl ring, pyridine ring, dimethylamino, sec.-propyl, the tertiary butyl.

3. the guanidine class NTR1 small molecular antagonists according to claims 1, is characterized in that, described substituted aroma ring is the phenyl ring or pyridine ring that are replaced by halogen, methyl or ethyl.

4. the guanidine class NTR1 small molecular antagonists according to claims 1, is characterized in that, described polar substitution group is amino, hydroxyl, aminomethyl or methylol.

5. the guanidine class NTR1 small molecular antagonists according to any one of claims 1 to 4, is characterized in that, the n in described general formula is 1-3.

6. the guanidine class NTR1 small molecular antagonists according to claims 1, it is characterized in that, described compound is preferably R ₁morpholine ring, R ₂contraposition chlorine substituted benzene ring, R ₃be hydrogen, n is 2, and its structure is as follows,

7. guanidine class NTR1 small molecular antagonists according to claim 1 is in preparation and the application in the medicine of NTR1 protein related diseases.