CN111686113B

CN111686113B - Sonic Hedgehog inhibitor and application thereof

Info

Publication number: CN111686113B
Application number: CN201910197092.XA
Authority: CN
Inventors: 刘莹; 来鲁华; 云泰康翔
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2022-12-30
Anticipated expiration: 2039-03-15
Also published as: CN111686113A

Abstract

The invention discloses a Sonic Hedgehog inhibitor and application thereof. The Sonic Hedgehog inhibitor is a pyrimidine compound, and is an ShhN inhibitor for inhibiting an Hh signal pathway through the verification of an in-vitro binding experiment and a cell test of the ShhN. The pyrimidine compound or the medicinal salt thereof is used as an effective component, and a conventional medicament carrier is added, so that medicaments for treating or preventing various diseases caused by improper regulation of an Hh signal pathway can be prepared, and the medicaments comprise various medicaments related to tumors.

Description

Sonic Hedgehog inhibitor and application thereof

Technical Field

The invention relates to a medicament for treating and preventing various diseases caused by improper regulation of a Hedgehog (Hh) signal pathway, in particular to a pyrimidine compound serving as a pathway upstream protein Sonic Hedgehog (Shh) inhibitor, and application of the compound and combined medicaments thereof in treating leukemia, basal cell carcinoma, pancreatic cancer, colon cancer, bladder cancer, small cell lung cancer and other diseases.

Background

The Hedgehog (Hh) signaling pathway plays an important role in cell differentiation, tissue development, and embryogenesis. In physiological conditions, the Hh signaling pathway is normally closed, but can be activated during tissue repair.

Aberrant activation of Hh signaling can lead to the formation of tumors, and about one-fourth of cancers are associated with inappropriate regulation of this pathway. The abnormal expression of Hh signaling pathway has been reported in bladder cancer, basal cell carcinoma, pancreatic cancer, colon cancer, medulloblastoma, prostate cancer, lung cancer, liver cancer, endometrial cancer, ovarian cancer, etc.

The antitumor drug is expected to be obtained by inhibiting the Hh signal channel. The Hh signaling pathway includes the targets of Sonic hedgehog (Shh), smoothened (Smo), and Glioma-associated family of translation factors (Gli). Studies with Smo as a drug target have resulted, and Smo antagonists are now approved by the FDA for clinical trials. The FDA approved Vismodegib (GDC-0449) for the treatment of basal cell carcinoma in adults in 2012; sonidegib (LDE 225) was approved for the treatment of advanced basal cell carcinoma in 2015. Smo antagonists in clinical trials are: LEQ506 (Phase I), LY2940680 (Phase II), IPI-926 (Phase II), BMS-833923 (XL-139) (Phase II), PF-04449913 (Phase II), and the like. However, in clinical trials, many small molecules have not shown inhibition of solid tumors (Gonnissen, et al, oncotarget,2015,6,13899), and there are reports of resistance of cancer cells to Vismodegib (Atwood, et al, journal of Cell Biology,2012,199,193). Inhibitor studies on Gli and Shh have not been reported to enter the clinical phase.

Shh is located upstream of the Hh signaling pathway, and the effect of suppressing Shh on the entire pathway is more pronounced. The human full-length Shh contains 462 amino acid residues in total, and consists of a signal peptide, an amino-terminal domain (Sonic hedgehog protein N-product, shhN) and a carboxyl-terminal domain (Sonic hedgehog protein C-product, shhC). Shh is autocatalytically cleaved in the endoplasmic reticulum to ShhN and ShhC. ShhN is considered as a drug target capable of avoiding toxic and side effects, and at present, only two non-antibody inhibitors for researching ShhN are reported, wherein one is cyclic peptide (Owens, et al, journal of the American Chemical Society,2017,139,12559) and the other is 12-membered cyclic organic small molecule with moderate activity (Stanton, et al, nature Chemical Biology,2009,5,154).

Disclosure of Invention

The invention aims to provide a pyrimidine compound as an inhibitor for inhibiting ShhN.

The invention also aims to provide the compound, which is applied to medicaments for treating and preventing various diseases caused by improper regulation of an Hh signal pathway. Abnormalities in Hh signaling pathways can lead to the formation of tumors, such as leukemia, bladder cancer, basal cell carcinoma, pancreatic cancer, colon cancer, medulloblastoma, prostate cancer, lung cancer, liver cancer, endometrial cancer, ovarian cancer, and the like, as well as other diseases.

The invention defines a binding interface with Patched (Ptch) based on the crystal structure of a complex of ShhN and an antibody and the like, and carries out rational drug design according to the interface. The method comprises the steps of carrying out virtual screening and manual selection on three representative compound libraries, finally selecting 209 compounds for experiment, and preliminarily obtaining the pyrimidine small molecule inhibitor acting on ShhN through Surface Plasmon Resonance (SPR) preliminary screening, micro-calorimetric swimming (MST) in-vitro target point verification, dual-luciferase reporter gene detection of an Hh signal path and target point verification on an RT-PCR cell level. The small molecules show high water solubility, low cytotoxicity and novel structure, and are subjected to analogue search based on the characteristics, and a plurality of analogues are purchased for intensive research to obtain a series of novel compounds with better activity on the cellular level and enhanced binding capacity.

The pyrimidine compound provided by the invention has the following structural general formula:

in the formula I, R represents hydrogen, -NHR ₁ A dialkylamino-substituted C2-C6 alkylmercapto group, a substituted tetrahydropyrazinyl group; wherein R is ₁ Represents hydrogen, morpholino C1-C6 alkyl, tetrahydrofuranyl C1-C6 alkyl, carboxylo C1-C6 alkyl, hydroxylo C1-C6 alkyl, C5-C7 cycloalkenylo C1-C6 alkyl, phenyl, substituted phenyl, C5-C7 cycloalkyl, diethylamino C1-C6 alkyl.

For R, the above-mentioned dialkylaminoc-C2-C6 alkylmercapto group is preferably a dialkylaminoc-C2-C3 alkylmercapto group, wherein the alkyl group is preferably a C1-C6 alkyl group, more preferably a C1-C3 alkyl group. The substituted tetrahydropyrazinyl group preferably has one or more C1-C6 alkyl substituents thereon, more preferably one or more C1-C3 alkyl substituents thereon.

For R ₁ Preferably represents hydrogen, morpholino C1-C3 alkyl, tetrahydrofuro C1-C3 alkyl, carboxyi C1-C3 alkyl, hydroxyi C1-C3 alkyl, C5-C7 cycloalkenylo C1-C3 alkyl, phenyl, substituted phenyl, C5-C7 cycloalkyl, diethylamino C1-C3 alkyl, wherein the substituted phenyl group may have one or more substituents, preferably halogen (F, cl, br, I) and/or C1-C6 alkoxy.

In formula I, W represents a carbon atom or a sulfur atom; t represents phenyl, substituted phenyl, 2-thienyl, -NR ₂ R ₃ (ii) a Q represents hydrogen, nitro, cyano, or with R ₃ The ring combination represents 1,2-pyrazolyl. Wherein R is ₂ 、R ₃ The same or different, each independently representHydrogen or one of the following groups:

wherein R is ₆ 、R ₇ 、R ₈ Each independently represents hydrogen, nitro, amino, halogen, C1-C6 alkyl, C1-C6 alkoxy; or R ₂ Represents phenyl, while R ₃ Cyclizing with Q represents 1,2-pyrazolyl.

In the formula I, R ₄ Represents hydrogen, phenyl, halogen-substituted phenyl, alkoxy-substituted phenyl, etc.

When T represents a substituted phenyl group, the substituted phenyl group has one or more substituents, preferably halogen (F, cl, br, I) and/or C1-C6 alkoxy.

When R is ₄ When an alkoxy-substituted phenyl group is represented, a substituted phenyl group having one or more C1-C6 alkoxy groups is preferred.

Specific examples of the above compounds of formula I are:

the pyrimidine compound is verified by ShhN in-vitro combination experiments and cell tests, and is a ShhN inhibitor for inhibiting an Hh signal channel.

The pyrimidine compound or the medicinal salt thereof is used as an effective component, and a conventional medicine carrier is added, so that the medicine for treating or preventing various diseases caused by improper regulation of an Hh signal pathway can be prepared, and the medicine comprises various tumor-related medicines.

The pharmaceutically acceptable salts of the pyrimidine compounds and the pharmaceutical compositions thereof of the present invention are pharmaceutically acceptable salts, such as salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, etc., or salts formed with organic acids such as citric acid, succinic acid, citric acid, acetic acid, tartaric acid, methanesulfonic acid, etc.

Conventional pharmaceutical carriers refer to nontoxic solid, semisolid, or liquid fillers, diluents, adjuvants, encapsulating materials, or other formulation adjuvants. The pharmaceutical composition may be formulated into various dosage forms according to the purpose of treatment, the need of administration route, according to the well-known technique in the art.

Drawings

FIG. 1 is a graph of the predicted binding pattern of the compound PKUMDL _ ShhN _001, wherein hydrogen bonds are indicated by arrows, salt bridges are indicated by lines, and pi-pi stacking is indicated by circular arrows.

Detailed Description

The following examples are intended to illustrate the invention and to describe in detail the process for practicing the invention without limiting the scope of the invention in any way. Those skilled in the art may find other ways of practicing the invention that are obvious to them and should be considered to be included within the scope of the invention.

Example 1 virtual screening of ShhN inhibitors

1. Exploration of ShhN binding sites

ShhN needs to interact with the transmembrane protein Ptch, conducting the signal downstream. At present, the crystal structure of the ShhN and Ptch complex is not solved. We found the crystal structure 3HO5 of the ShhN and hedgehog-interacting protein (HHIP) binary compound and the crystal structure 3MXW of the ShhN and 5E1fab fragment binary compound, and searched the crystal structures of 3HO5 and 3MXW with a distance of 5E1fab fragment or HHIP

The relevant ShhN residues are marked in the crystal structure 3M1N of ShhNThe residues serve as the protein-protein interaction interface for ShhN.

2. Virtual screening of compounds based on interaction interface between ShhN and Ptch protein-protein

Based on the protein-protein interaction interface of ShhN, we considered SPECS Library (11 month version 2014, approximately 200000 compounds) and Maybridge Screening Library (2015 5 month version, approximately 50000 compounds) as well as the Life Chemicals PPIInhibitors 3D Library (2015 2 month version, approximately 17000 compounds) built specifically for the development of protein-protein interaction (PPI) inhibitors, and conducted targeted virtual Screening.

First, a first round of rough docking was performed using Glide SP, and compounds 5% of Glide SP Score in front of each compound library were selected for the next round of screening. Then adopting a Glide XP mode to perform further virtual screening. 318 compounds with Glide XP Score less than-6.0 kcal/mol are selected for prediction of the properties of the general screening interference compounds (PAINS). The 263 compounds that passed the prediction were manually selected and finally 209 compounds were selected and purchased for subsequent experimental studies.

3. ShhN inhibitor docking

Taking PKUMDL _ ShhN _001 as an example, the interaction between the small molecule and ShhN is analyzed. The furan ring of PKUMDL _ ShhN _001 forms pi-pi interaction with His134, his140 and His182, respectively, one imino group forms a hydrogen bond with the side chain oxygen atom of Asp147, and two salt bridges are formed between proteins (see FIG. 1).

For the purchased PKUMDL _ ShhN _001, utilize ¹ The structural correctness was verified by H NMR and high resolution mass spectrometry: ¹ H NMR(DMSO-d6,400MHz):δ9.51(dt,J＝69.8,5.6Hz,1H),8.50(d,J＝70.8Hz,1H),7.91(d,J＝122.7Hz,1H),7.58(s,1H),7.48(m,1H),6.35(m,2H),4.67(dd,J＝12.8,5.6Hz,2H),3.55(m,4H),3.28(m,2H),2.30(m,6H),1.64(tt,J＝13.9,7.0Hz,2H).HRMS(ESI):calcd for C16H23N7O4,[(M+H)+],378.1884,found 378.1885.

designing an inhibitor shown as the following formula I through the molecular docking result:

r, W, Q, T and R in formula I ₄ The groups represented are as described previously.

4. Analog search

1.2D analog search

A total of 45 molecules from Life Chemicals PPIInhibitors 3D Similarity Library share greater than 65% Similarity to PKUMDL _ ShhN _ 001. Several molecules were purchased by manual screening.

2.3D analog search

Life Chemicals PPIInhibitors 3D Similarity Library and SPECS Library were selected. In Life Chemicals PPIInhibitors 3D Similarity Library, a Shape Sim score of greater than 0.45 for a total of 44 molecules was manually screened to purchase several molecules. In SPECS Library, a total of 26 molecules with a Shape Sim score greater than 0.60 were purchased for several molecules by manual screening.

PKUMDL _ ShhN _015 occupies a similar space as 3D of PKUMDL _ ShhN _001, but a condensed ring and a halogen-containing benzene ring at the center of PKUMDL _ ShhN _015 make it more hydrophobic. The PKUMDL _ ShhN _015 with strong lipophilicity and large volume better conforms to the tendency characteristic of a PPI inhibitor, and is expected to become a potential ShhN inhibitor.

Example 2 Experimental validation of ShhN inhibitors

1. Construction of ShhN plasmid and purification expression of protein

The information on the protein expression plasmid is as follows: the expression vector is pET-28a, the segment connected with the Shh is 24-197aa, and the fusion protein is N-his-ShhN.

An expression step: the plasmid was transformed into E.coli BL21 competent cells. 50. Mu.L of 30mg/mL Kan was added to 50mL of LB medium, and 1mL of the strain was inoculated. The culture was carried out overnight at 37 ℃ and 220 rpm. 1L LB medium was added with 1mL 30mg/mL Kan, then 10mL of the cultured broth was inoculated, and OD600 to 0.6 was cultured at 37 ℃ and 220 rpm. The temperature was lowered to 25 ℃ and 0.8mL of 1M IPTG was added to the 1L culture medium and cultured for 8 hours. Pouring the bacterial liquid into a centrifugal cup, balancing, centrifuging at 6000rpm for 10min, discarding a supernatant, and freezing bacterial mud to-80 ℃ for later use.

A purification step: the sludge was thawed by stirring and resuspended in lysis buffer (20mM HEPES, pH 7.4, 300mM NaCl,0.1mM PMSF,1mM DDT). Sonication in ice bath (3 s pulses, 5s intervals, 99 sonications at 400W) was repeated 3 times. The lysate was poured into centrifuge tubes, trimmed, and centrifuged at 17000rpm for 30min at 4 ℃. The supernatant was aspirated and filtered through a 0.22 μm pore size filter.

The A, B pump of FPLC was flushed with Buffer A (20mM HEPES, pH 7.4, 300mM NaCl,40mM imidazole), buffer B (20mM HEPES, pH 7.4, 300mM NaCl,500mM imidazole), respectively. Histrap FF5mL column was equilibrated with Buffer A. The supernatant was loaded at 1mL/min and washed with 3mL/min Buffer A until the UV returned to baseline, approximately 30 column volumes. Elution was carried out with a 0-100% buffer B gradient at 3mL/min over 20 min. The eluted peak was collected and concentrated to 2mL.

The sample was applied to a Sephacryl S-200 gel column equilibrated with Buffer C (20mM HEPES, pH 7.4, 300mM NaCl) and eluted at 1mL/min Buffer C.

The collected ShhN protein was concentrated, dispensed with PCR tubes, snap frozen in liquid nitrogen and stored at-80 ℃.

2. Construction of ShhN-EGFP plasmid

First, the plasmid pET-28a containing ShhN and the plasmid pEGFP-N1 containing EGFP were treated with HindIII and NotI enzymes, respectively. The linearized pET-28a plasmid is then ligated with the gene of interest EGFP using T4DNA ligase. Due to the frame shift, 1 extra base needs to be mutated by PCR. The designed primers are as follows:

5'-TCCACCGGTCGCCACATGGTGAGCAAGGGCGAG-3'(SEQ ID No：1)；

5'-CTCGCCCTTGCTCACCATGTGGCGACCGGTGGA-3'(SEQ ID No：2)。

after mutation, transformation and sequencing are carried out, and the pET-28a plasmid containing ShhN-EGFP is successfully constructed.

And (4) carrying out expression and purification of the ShhN-EGFP protein by referring to the method in the previous step.

3. Surface Plasmon Resonance (SPR) screening

The protein of ShhN was attached to the chip surface of SPR. The instrument used in this experiment was Biacore T200. ShhN was diluted to 10 ng/. Mu.L with 10mM sodium acetate, pH 5.5. Approximately 1000RU protein was immobilized on CM5 chips at 25 ℃ in a standard EDC/NHS amino coupling format. The reference lane was activated as a blank reference. 10mM HEPES, pH 7.4, 150mM NaCl,0.05% of the mobile phase and the loading solution in DMSO. The analytical data Software was Biacore T200Evaluation Software. Small molecules with RU greater than 5.0 at 50. Mu.M were further studied.

4. Ligand binding assays based on microcalorie thermoloresis (MST)

In the experiment, the ShhN fusion protein containing EGFP is adopted and is not marked by fluorescent dye, so that the influence of the dye on the protein in the marking process can be avoided.

The buffer composition of MST was 50mM Tris-HCl, pH 7.4, 150mM NaCl,10mM MgCl ₂ 0.1% Tween-20,0.1% Pluronic-F127,5% DMSO; set the instrument conditions to blue LED light source, 20% LED Power and 40% MST Power. During the experiment, the concentration of ShhN-EGFP is kept constant, and micromolecule solutions with different concentrations and equal volumes are added. Nanotemper Analysis software V1.5.41 was used for data fitting. The results are shown in Table 1, compound K _d Below 10. Mu.M, a good binding force to ShhN is exhibited.

5. Cell assay of Compounds

By detecting ptch by RT-PCR ^+/- p53 ^-/- Determining the action target of the compound according to the mRNA transcription level of the Gli1 of the medulloblastoma cells. The experiment shows that: (1) Positive reference compound Robotnikin targeting ShhN on ptch ^+/- p53 ^-/- mRNA expression of the glioblastoma cell Gli1 had no effect; (2) Small molecule compound GDC-0449 targeting key signal molecule Smo downstream of Ptch can significantly inhibit Ptch ^+/- p53 ^-/- mRNA transcription of medulloblastoma cell Gli 1; (3) Compound PKUMDL _ ShhN _001 vs ptch ^+/- p53 ^-/- mRNA transcription levels of the medulloblastoma cell Gli1 were not significantly affected; the results suggest that the test compound PKUMDL _ ShhN _001 might act on the Ptch upstream key signal molecule Shh.

Similar compounds are screened by using a dual-luciferase reporter gene experiment, except that the compounds PKUMDL _ ShhN _012, PKUMDL _ ShhN _017 and PKUMDL _ ShhN _018 have high toxicity, and the inhibition effect on an Hh signal channel cannot be accurately detected, and most of the rest analogues have activity.

Further IC was determined for these analogous compounds ₅₀ . Table 1 shows the results of experiments with 10 compounds, PKUMDL _ ShhN _001 (IC) ₅₀ =2.2 ± 0.3 μ M) in comparison: (1) In the general formula R ₃ And Q forms a ring, R ₄ Has improved activity when it is benzene ring, and has condensed skeleton, and has 3 compounds including PKUMDL _ ShhN _014, PKUMDL _ ShhN _015 and PKUMDL _ ShhN _016, and IC of PKUMDL _ ShhN _0015 ₅₀ Reaching 0.4 mu M, and the activity is 5 times higher than that of PKUMDL _ ShhN _ 001; (2) The R position in the general formula is replaced and the activity is equivalent or reduced, such as PKUMDL _ ShhN _004, PKUMDL _ ShhN _007, PKUMDL _ ShhN _008 and PKUMDL _ ShhN _009 show equivalent activity, and the activity of PKUMDL _ ShhN _003 and PKUMDL _ ShhN _010 is reduced in a small degree; (3) Q is cyano, R ₃ PKUMDL _ ShhN _022 activity, which is chlorophenyl, was increased.

TABLE 1 Experimental results for representative ShhN inhibitors

^a mean±S.D.,n＝3.

The PKUMDL _ ShhN _001 and the analogues are screened, and the in vitro combination and cell experiments prove that the PKUMDL _ ShhN _001 and the analogues act on ShhN targets preliminarily, two types of compounds with patent drug potential are obtained, one type is a pyrazolopyrimidine fused ring compound, and the most obvious activity improvement is PKUMDL _ ShhN _015 (IC) ₅₀ =0.4 ± 0.2 μ Μ); another class is pyrimidine monocyclic compounds T, Q which are both different from PKUMDL _ ShhN _001, such as PKUMDL _ ShhN _022 (IC) ₅₀ ＝1.2±0.2μM)。

The test results show that the compound has the functions of inhibiting ShhN and regulating the Hh signal channel. The pyrimidine compound or the medicinal salt thereof is used as an effective component, and a conventional medicine carrier is added to prepare medicines for treating or preventing various tumors.

The pharmaceutically acceptable salts of the pyrimidine compounds of the present invention are pharmaceutically acceptable salts, for example, salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and nitric acid, or salts formed with organic acids such as citric acid, succinic acid, citric acid, acetic acid, tartaric acid, and methanesulfonic acid.

SEQUENCE LISTING

<110> Beijing university

<120> Sonic Hedgehog inhibitor and application thereof

<130> WX2019-03-042

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 33

<212> DNA

<213> Artificial sequence

<400> 1

tccaccggtc gccacatggt gagcaagggc gag 33

<210> 2

<211> 33

<212> DNA

<213> Artificial sequence

<400> 2

ctcgcccttg ctcaccatgt ggcgaccggt gga 33

Claims

1. Use of a pyrimidine compound, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of cancer caused by dysregulation of the Hh signaling pathway, wherein the pyrimidine compound is selected from one of the following compounds PKUMDL _ ShhN _001 to PKUMDL _ ShhN _ 023:

2. the use of claim 1, wherein the cancer is leukemia, bladder cancer, basal cell carcinoma, pancreatic cancer, colon cancer, medulloblastoma, prostate cancer, lung cancer, liver cancer, endometrial cancer, ovarian cancer.

3. Use of the pyrimidine compound or its pharmaceutically acceptable salt according to claim 1 for the production of ShhN inhibitors.