CN112028884A - Novel organic compound and preparation method and application thereof - Google Patents
Novel organic compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a novel organic compound, a preparation method and application thereof, wherein molecular dynamics is used for simulating the interaction between c1-c8 and a ryanodine receptor, the strength of the interaction is evaluated by using a free energy calculation and alanine scanning method, and a pharmacophore of a ryanodine receptor inhibitor c8 is discovered by using a combined calculation method. Finally, a compound (compound d1-d11) with a novel structure is synthesized based on the pharmacophores, and the compound has good biological activity on staphylococcus aureus and influenza virus, greatly enhances the bactericidal activity on staphylococcus aureus, has an inhibiting effect on influenza virus, and has a good development prospect.
Description
Technical Field
The invention relates to the technical field of drug development, in particular to a novel organic compound and a preparation method and application thereof.
Background
Ryanodine receptors (RyR) are widely distributed in muscle tissue of mammals, insects, and other species. They are the largest known ion channel proteins, consisting of four substructures containing about 5000 amino acid residues. Each subunit comprises a cytoplasmic domain and a transmembrane domain. Its main function is to regulate the concentration of calcium ions in the cytoplasm and to control muscle contraction and relaxation, and when the ryanodine receptor is inhibited by a ligand, the imbalance in the concentration of calcium ions in the cytoplasm results in failure of the muscle to contract and relax normally. RyR1 is one of the ryanodine receptors, and is found primarily in skeletal muscle. Chlorantraniliprole belongs to bisamide compounds, is a widely used insecticide at present, and has an action target of a ryanodine receptor. Some bisamide compounds (e.g., compounds c1-c8 described herein) are capable of malfunctioning the ryanodine receptor (Lahm et al, 2005). Compound c1-c8 was considered to act on the hotspot ring of RyR1, as in fig. 17a (Amador et al, 2009). This region is called a hot spot loop because mutations in amino acid residues in the loop can lead to disease.
On the other hand, drug development is a time consuming and expensive task. Developing a large summary of marketed drugs takes 10-15 years and 5-8 billion dollars. Computer-assisted drug design is widely used in pharmaceutical enterprises to speed this process. Computer-aided drug design helps scientists focus on the most potential compounds for development, thus minimizing the cost of compound synthesis and bioactivity testing. The choice of computer-aided drug design methodology in practice depends on the 3D structure of the protein to be studied. Ligand-based drug design approaches such as Quantitative Structure Activity Relationship (QSAR) and pharmacophore analysis are generally chosen when the structure of the protein is ambiguous. When the structure of the target protein for drug action is known, receptor-based drug design methods, such as molecular docking, can be used. The invention constructs an ideal model based on theoretical calculation and develops a new compound with expected high activity by using the model.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a novel organic compound which can be used for preparing medicines for inhibiting staphylococcus aureus and influenza virus.
The second object of the present invention is to provide a process for producing a novel organic compound.
It is a further object of the present invention to provide a pharmaceutical which can be synthesized from the above novel organic compound.
The fourth purpose of the invention is to provide a preparation method of the medicine.
The fifth purpose of the invention is to use the medicine or the pharmaceutically acceptable salt thereof in inhibiting staphylococcus aureus and influenza virus.
One of the purposes of the invention is realized by adopting the following technical scheme:
a novel organic compound having the structure shown in formula I:
in the formula I, R1Is H or halogen.
Further, the structure of the novel organic compound is shown as a formula II or a formula III:
the second purpose of the invention is realized by adopting the following technical scheme:
a preparation method of a novel organic compound is disclosed, wherein when the structure of the novel organic compound is shown as a formula II, the preparation method comprises the following steps:
compound d1 preparation procedure: dissolving Compound A in SOCl2And refluxing the excess SOCl2Removing the residues by vacuum pumping, dissolving the residues in pyridine, adding methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, concentrating the mixture under reduced pressure, extracting the residues with water and ethyl acetate, washing an organic phase with brine, drying the organic phase with anhydrous sodium sulfate, evaporating the organic solvent after drying, and purifying the residues by silica gel column chromatography to obtain a compound shown as a formula II, wherein the compound is marked as a compound d 1;
when the structure of the novel organic compound is shown as a formula III, the preparation method comprises the following steps:
compound d1 preparation procedure: dissolving the compound A inIn SOCl2And refluxing the excess SOCl2Vacuum pumping to remove residues, dissolving the residues in pyridine, adding methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, then concentrating the mixture under reduced pressure, extracting the residues with water and ethyl acetate, washing an organic phase with brine, drying the organic phase with anhydrous sodium sulfate, evaporating to remove an organic solvent after drying, and purifying the residues by using a silica gel column chromatography to obtain a compound d1 shown in formula II;
compound d2 preparation procedure: dissolving a compound d1 in chloroform, adding nitrogen bromosuccinimide dissolved in chloroform, then distilling the mixture under reduced pressure, and carrying out silica gel column chromatography on the residue to obtain a compound shown as a formula III, wherein the compound is marked as a compound d 2;
the structural formula of the compound A is shown as follows:
further, in the preparation step of the compound d1, 0.15g of the compound A was dissolved in 10mL of SOCl2And refluxed for 1 hour for excess SOCl2Removing by vacuum pumping, dissolving the residue in 5mL of pyridine, adding 0.5mmol of methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, concentrating the mixture under reduced pressure after 1 hour, extracting the residue with 5mL of water and 20mL of ethyl acetate, washing an organic phase with 5mL of brine, drying with anhydrous sodium sulfate, evaporating to remove the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound d1 shown in the formula II;
in the preparation step of the compound d2, 0.22g of the compound d1 is dissolved in 20mL of chloroform, 0.6mmol of nitrogen bromosuccinimide dissolved in chloroform is added, after 10 hours, the mixture is distilled under reduced pressure, and the residue is subjected to silica gel column chromatography to obtain the compound d2 shown in the formula III.
The third purpose of the invention is realized by adopting the following technical scheme:
a medicine is prepared from the novel organic compound, and the structure of the medicine is shown as a formula IV:
in the formula IV, R2Is H, alkyl, alkoxy, -CF3One of halogen or nitro.
Further, the structures of the medicines are shown as formulas V to XIII, which are respectively marked as compounds d3 to d 11:
the fourth purpose of the invention is realized by adopting the following technical scheme:
a method for preparing a medicament, the method for preparing compounds d3-d11 comprises the following steps:
compound d1 preparation procedure: dissolving Compound A in SOCl2And refluxing the excess SOCl2Vacuum pumping to remove residues, dissolving the residues in pyridine, adding methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, then concentrating the mixture under reduced pressure, extracting the residues with water and ethyl acetate, washing an organic phase with brine, drying the organic phase with anhydrous sodium sulfate, evaporating to remove an organic solvent after drying, and purifying the residues by using a silica gel column chromatography to obtain a compound d1 shown in formula II;
compound d2 preparation procedure: dissolving a compound d1 in chloroform, adding nitrogen bromosuccinimide dissolved in chloroform, then distilling the mixture under reduced pressure, and carrying out silica gel column chromatography on the residue to obtain a compound d2 shown in a formula III;
preparation steps of compounds d3-d 11: dissolving the compound d2 in a mixture of triethanolamine and dimethylformamide, and then adding the predetermined alkyne derivative, cuprous iodide and PdCl2(PPh3)2Adding the mixture into the solution, heating and stirring, cooling the mixture to room temperature, filtering the mixture by using kieselguhr, washing the kieselguhr by using ethyl acetate, concentrating the washing liquor under reduced pressure, extracting the residue by using water and ethyl acetate, washing an organic phase by using brine, drying the organic phase by using anhydrous sodium sulfate, vacuumizing the organic solvent, and purifying the mixture by using a silica gel column chromatography to obtain compounds d3-d11 shown in formulas V-XIII;
the structural formula of the compound A is shown as follows:
further, the preparation method of the compounds d3-d11 comprises the following steps:
compound d1 preparation procedure: 0.15g of Compound A was dissolved in 10mL of SOCl2And refluxed for 1 hour for excess SOCl2Removing by vacuum pumping, dissolving the residue in 5mL of pyridine, adding 0.5mmol of methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, concentrating the mixture under reduced pressure after 1 hour, extracting the residue with 5mL of water and 20mL of ethyl acetate, washing an organic phase with 5mL of brine, drying with anhydrous sodium sulfate, evaporating to remove the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound d1 shown in the formula II;
compound d2 preparation procedure: dissolving 0.22g of compound d1 in 20mL of chloroform, adding 0.6mmol of nitrogen bromosuccinimide dissolved in chloroform, distilling the mixture under reduced pressure after 10 hours, and performing silica gel column chromatography on the residue to obtain a compound d2 shown in the formula III;
preparation steps of compounds d3-d 11: dissolving 0.5mmol of compound d2 in 2mL of a mixture of triethanolamine and dimethylformamide in a volume ratio of 1: 1; 0.5mmol of the predetermined alkyne derivative, 0.05mmol of cuprous iodide and 0.05mmol of PdCl2(PPh3)2Adding into the solution, stirring at 85 deg.C for 12 hr, cooling the mixture to room temperature, filtering with diatomite, washing with ethyl acetate, concentrating the washing solution under reduced pressure, extracting the residue with 3ml water and 10ml ethyl acetate, washing the organic phase with 5ml brine, drying with anhydrous sodium sulfate, vacuum pumping off the organic solvent, and purifying the mixture with silica gel column chromatography to obtain compounds d3-d11 shown in formulas V-XIII.
Further, in the preparation steps of compounds d3-d11, when the drug is compounds d3-d11, respectively, the predetermined alkyne derivative is compounds b2-b 10:
the fifth purpose of the invention is realized by adopting the following technical scheme:
the application of the medicine or the pharmaceutically acceptable salt thereof in inhibiting staphylococcus aureus and influenza virus.
Compared with the prior art, the invention has the beneficial effects that:
the novel structural compound (the compound d3-d11 shown in the formula V-XIII) has good biological activity on Staphylococcus aureus (Staphylococcus aureus) and influenza virus, greatly enhances the bactericidal activity on Staphylococcus aureus, has an inhibiting effect on influenza virus, and has good development prospect.
Drawings
FIG. 1 is a graph of the molecular coordinates of a ryanodine receptor inhibitor c1 after geometric optimization, provided by an embodiment of the present invention;
FIG. 2 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c1 molecule provided by an embodiment of the present invention;
FIG. 3 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c2 after geometric optimization as provided by the present invention;
FIG. 4 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c2 molecule provided by an embodiment of the present invention;
FIG. 5 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c3 after geometric optimization as provided by the examples of the present invention;
FIG. 6 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c3 molecule provided by an embodiment of the present invention;
FIG. 7 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c4 after geometric optimization as provided by the examples of the present invention;
FIG. 8 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c4 molecule provided by an embodiment of the present invention;
FIG. 9 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c5 after geometric optimization as provided by the examples of the present invention;
FIG. 10 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c5 molecule provided by an embodiment of the present invention;
FIG. 11 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c6 after geometric optimization as provided by the examples of the present invention;
FIG. 12 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c6 molecule provided by an embodiment of the present invention;
FIG. 13 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c7 after geometric optimization as provided by the examples of the present invention;
FIG. 14 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c7 molecule provided by an embodiment of the present invention;
FIG. 15 is a graph of the molecular coordinates of the ryanodine receptor inhibitor c8 after geometric optimization as provided by the examples of the present invention;
FIG. 16 is a graph of the kinetic simulation of the ryanodine receptor inhibitor c8 molecule provided by an embodiment of the present invention;
FIG. 17 is a general flow chart for studying the interaction of RyR1 and c8 based on molecular force field and quantum mechanical methods provided by embodiments of the present invention;
FIG. 18 is a graph of the kinetic equilibrium in a molecular docking simulation provided by an embodiment of the present invention;
FIG. 19 is an atomic diagram of NBO analysis provided by embodiments of the present invention.
FIG. 20 is a flow chart of the design of new photosensitizers based on the pharmacophores of b1 and c8 provided by the present invention;
FIG. 21 is a diagram of a synthetic pathway for compounds d1-d11 provided by an example of the present invention;
FIG. 22 is a single crystal structure of compound d8 provided in the examples of the present invention to clarify the conformation of the synthesized molecules.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict. The following are specific examples of the present invention, and raw materials, equipments and the like used in the following examples can be obtained by purchasing them unless otherwise specified.
A series of bisamide compounds (c1-c8) are found to enable ryanodine receptors to release calcium ions uncontrollably to influence the activities of lepidopteran insects. The invention uses molecular simulation technology to simulate the interaction between RyR1 and inhibitor, hopes to simulate the interaction between bisamide compounds and ryanodine receptor and deduces the pharmacophore through a combined calculation method on the basis. The present invention uses molecular dynamics to mimic the interaction of c1-c8 with the ryanodine receptor. The strength of this interaction was evaluated using free energy calculations and alanine scanning methods. Natural bond orbital analysis and methods of atoms in molecules are used to analyze the interaction between the ryanodine receptor 1, RyR1 and its inhibitors. In order to investigate the pharmacophore of the bisamide type ryanodine receptor inhibitor, the invention uses a plurality of molecular simulation techniques to simulate the interaction of the compound c8 and RyR1, and discovers the pharmacophore of the ryanodine receptor inhibitor c8 by using a combined calculation method. Finally, compounds with novel structures (compounds d1-d11 shown in formulas I-XIII) are synthesized based on the pharmacophores, and the novel compounds have good activity of killing staphylococcus aureus and influenza virus. Specific embodiments are described below.
Simulation method
1.1 molecular docking and molecular dynamics simulation
1.1.1 computing resources
W580i desktop supercomputer (with four NVIDIAC2050 computing cards, counting 1 trillion times per second per card): zhongke eosin.
1.1.2 calculation software
AutoDock software package: downloaded from http:// autodock. script. edu/downloads;
AMBER software package: AMBER software Inc., USA;
PyMOL software: delano Scientific LLC, USA;
VMD software package: from http:// www.ks.uiuc.edu/Research/vmd/download;
discovery Studio 3.0 Client: accrys, usa.
1.1.3 calculation method
The binding process of the ryanodine receptor inhibitors c1-c8 and the RyR1 hot spot loop (PDB No. 3HSM) was simulated using the AutoDock software package, respectively. The same approach was used to mimic the hot-spot loop binding process of the homologous proteins Drosophila melanogaster (Drosophila simulans) of the rybut receptor inhibitors c1-c8 and RyR 1. The calculation uses the lamark genetic algorithm. The coordinates generated by the AutoDock software package are compared to the geometrically optimized coordinates. The coordinate with the least root mean square deviation between the two is considered as a reasonable initial coordinate for the kinetic calculations.
The complex of reasonable coordinates and RyR1 or a homologous model serves as the initial coordinates for kinetic calculations. All molecular dynamics simulations used the AMBER software package. TIP3P solvent water was used for the simulation. Sodium ions are used to balance the negative charge in the mimetic system making it electrically neutral. This mimetic system includes a receptor, a ligand, approximately 7800 water molecules, and 7 sodium ions. The calculation uses the AMBER force field and the ff99SB force field. The ligand uses the RESP charge. A usage period boundary condition is calculated. The energy of the system is minimized, firstly, 2000 steps are calculated by using a steepest descent method, and then 2000 steps are calculated by using a conjugate gradient method. The energy minimized composite was heated to 50ps prior to density equilibration to 50ps followed by 300 kelvin and a pressure equilibration of 500ps at one standard atmosphere, and finally a 2ns molecular dynamics simulation was performed.
Mpi module in AMBER software package is used for energy minimization, heating and density balancing in the computing system. Pressure balance and molecular dynamics simulations used the pmemd.cuda module in the AMBER software package. The analysis of the root mean square deviation uses the ptraj module in the AMBER software package. The MM-GBSA module in the AMBER software package is used in conjunction with the calculation of the free energy.
All calculations are based on simulated atomic jitter. The step size is 2 fs. The temperature was controlled using langevin kinetics. The NPT family was used in pressure equilibrium and 2ns molecular dynamics simulations.
The calculation of the binding free energy (Δ Gbind) for each inhibitor uses the equation:
ΔGbind＝Gcom–Grec–Glig (1)
com, rec and lig in the equation represent the complex, receptor and ligand, respectively. Their free energy is affected by four aspects:
G＝<EMM>+<Gpsolv>+<Gnpsolv>-T<S> (2)
EMMis the molecular force field energy, which represents the molecular internal energy, static electricity and van der waals interactions. Gpsolv represents the polar contribution in the polarization energy of the molecule. Gnpsolv represents a non-polar solvation energy. T represents the absolute temperature and S represents the entropy of the molecule.
The nonpolar solvation term Gnpsolv is calculated by solvating surface area (SASA) using the equation:
Gnpsolv＝γSASA+b (3)
SASA uses Molsurf method with a probe radius of
Using GB polar solvation energy of AMBER
b＝0kcal/mol。
Entropy change of solute nmode module using AMBER was calculated using a general frequency analysis method. The calculation of entropy uses complexes of whole proteins and ligands. In the dynamic simulation, one coordinate is intercepted every 10fs, and in the dynamic simulation of 2000fs, a total of 200 coordinates are intercepted and used for calculating the MM-GBSA. The energy calculation uses the mm-gbsa module in AMBER. The internal, electrostatic and van der waals energies were calculated using the sander module without interrupting the non-bond interactions. Polar solvation free energy Gpsolv was achieved using the GB method in AMBER 11.
Alanine scanning is performed by substituting the amino acid residue on the side chain with alanine, and then calculating the binding free energy of the mutant system. The difference between wildness and variants, Δ Gbind, can be compared to mutation tests:
ΔGbind＝ΔGbind mutant-ΔGbind wildtype (4)
the binding free energy of the alanine mutants was calculated using the method of MM-GBSA. Complex coordinates were calculated using wild type truncation.
Bond interactions can be described as Δ E by the free energy of stabilization(2). It uses a second order perturbation analysis Fock matrix, which is obtained by NBO analysis. By this perturbation method, a filled-in-orbit sigma (donor) and an empty-in-orbit can be quantitatively described
(acceptor) interaction, the stabilizing free energy can be calculated by the formula:
f is the Fock operator.
And
NBO energy donor and acceptor orbitals, respectively.
Single point analysis was performed using the energy-lowest conformation of c8 and RyR1 in a molecular dynamics simulation of 20 ns. Every 10fs, 1 coordinate is truncated, for a total of 2000 calculated coordinates. Methods of analyzing atoms in molecules are used. The calculations use the AIM2000 software package. Comparing the results of atom analysis in the molecule with the results of natural bond orbital analysis, and mutually verifying.
1.2 results and analysis
1.2.1 Complex conformation by molecular docking
The present invention compares the geometrically optimized coordinates of the ryanodine receptor inhibitors c1-c8 (tables 1, 3, 5, 7, 9, 11, 13 and 15) with those obtained after docking to yield the possible conformations of the c1-c8 molecules when bound to ryanodine receptors (tables 2, 4, 6, 8, 12, 14, 16). The full atomic root mean square deviation of these initial and geometrically optimized coordinates for molecular dynamics calculations has a minimum compared to other conformations generated by docking (table 17). The calculations of the present invention can be repeated using the possible conformations provided by the present invention (table 2, table 4, table 6, table 8, table 12, table 14, table 16). As shown in FIGS. 1-16, there are graphs of ryanodine receptor inhibitors c1-c8, respectively.
TABLE 1 molecular coordinates of the ryanodine receptor inhibitor c1 after geometric optimization
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N | 1.210 | 1.479 | 0.226 | 24 | C | -1.240 | 0.082 | -0.923 |
| 2 | C | 0.043 | 2.082 | -0.149 | 25 | C | -4.819 | -1.051 | -0.457 |
| 3 | O | 0.036 | 3.261 | -0.525 | 26 | F | -4.951 | -1.699 | -1.644 |
| 4 | H | 2.031 | 2.058 | 0.084 | 27 | F | -4.851 | -2.020 | 0.506 |
| 5 | C | 1.398 | 0.203 | 0.841 | 28 | F | -5.944 | -0.312 | -0.285 |
| 6 | C | 2.237 | -0.747 | 0.231 | 29 | H | 3.095 | -2.716 | 0.395 |
| 7 | C | 2.453 | -1.978 | 0.865 | 30 | H | 1.998 | -3.233 | 2.555 |
| 8 | C | 1.83 | -2.271 | 2.080 | 31 | H | 0.516 | -1.540 | 3.629 |
| 9 | C | 0.996 | -1.324 | 2.680 | 32 | H | 0.173 | 0.665 | 2.547 |
| 10 | C | 0.794 | -0.085 | 2.070 | 33 | H | 4.635 | -0.161 | 0.697 |
| 11 | C | 2.786 | -0.511 | -1.157 | 34 | H | 6.929 | -1.131 | 0.437 |
| 12 | O | 2.017 | -0.560 | -2.133 | 35 | H | 6.683 | -1.252 | -1.314 |
| 13 | N | 4.110 | -0.287 | -1.300 | 36 | H | 5.691 | -2.227 | -0.210 |
| 14 | H | 4.407 | -0.179 | -2.266 | 37 | H | 6.559 | 1.404 | 0.364 |
| 15 | C | 5.146 | -0.115 | -0.267 | 38 | H | 5.063 | 2.063 | -0.326 |
| 16 | C | 6.172 | -1.253 | -0.344 | 39 | H | 6.300 | 1.357 | -1.387 |
| 17 | C | 5.803 | 1.263 | -0.414 | 40 | H | -4.443 | 1.263 | 0.896 |
| 18 | C | -1.218 | 1.258 | -0.158 | 41 | H | -3.288 | 2.997 | 1.970 |
| 19 | C | -2.380 | 1.713 | 0.501 | 42 | H | -2.46 | 3.863 | 0.666 |
| 20 | C | -3.542 | 0.940 | 0.383 | 43 | H | -1.519 | 3.097 | 1.939 |
| 21 | C | -2.412 | 2.984 | 1.317 | 44 | H | -2.424 | -1.562 | -1.651 |
| 22 | C | -3.564 | -0.226 | -0.387 | 45 | H | -0.336 | -0.246 | -1.429 |
| 23 | C | -2.412 | -0.66 | -1.048 |
Table 2 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c1
TABLE 3 molecular coordinates of the ryanodine receptor inhibitor c2 after geometric optimization
Table 4 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c2
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N1 | 0.401 | 3.163 | 62.741 | 26 | C19 | -0.737 | 7.352 | 60.232 |
| 2 | C1 | 0.386 | 4.532 | 62.700 | 27 | C20 | 4.592 | 7.519 | 60.201 |
| 3 | O1 | -0.539 | 5.163 | 63.209 | 28 | F1 | 4.074 | 8.172 | 59.138 |
| 4 | H1 | -0.361 | 2.783 | 63.293 | 29 | F2 | 5.615 | 6.76 | 59.751 |
| 5 | C2 | 1.336 | 2.232 | 62.185 | 30 | F3 | 5.116 | 8.457 | 61.021 |
| 6 | C3 | 2.218 | 1.563 | 63.055 | 31 | H3 | 3.78 | 0.079 | 63.219 |
| 7 | C4 | 3.099 | 0.602 | 62.548 | 32 | H4 | 3.805 | -0.421 | 60.784 |
| 8 | C5 | 3.106 | 0.314 | 61.184 | 33 | H5 | 2.231 | 0.734 | 59.269 |
| 9 | C6 | 2.218 | 0.968 | 60.333 | 34 | H6 | 0.485 | 2.182 | 58.859 |
| 10 | C7 | 1.308 | 1.920 | 60.812 | 35 | H7 | 0.483 | 3.648 | 59.867 |
| 11 | C8 | 0.329 | 2.569 | 59.866 | 36 | H8 | -0.689 | 2.347 | 60.187 |
| 12 | C9 | 2.317 | 1.973 | 64.505 | 37 | H9 | 0.865 | -0.314 | 64.279 |
| 13 | O2 | 3.021 | 2.940 | 64.829 | 38 | H10 | 0.065 | -1.678 | 66.182 |
| 14 | N2 | 1.661 | 1.232 | 65.427 | 39 | H11 | 0.649 | -0.432 | 67.310 |
| 15 | H2 | 1.809 | 1.550 | 66.380 | 40 | H12 | 1.806 | -1.329 | 66.298 |
| 16 | C10 | 0.668 | 0.165 | 65.238 | 41 | H13 | -1.473 | -0.036 | 65.049 |
| 17 | C11 | 0.807 | -0.895 | 66.336 | 42 | H14 | -0.814 | 1.467 | 64.359 |
| 18 | C12 | -0.745 | 0.763 | 65.189 | 43 | H15 | -0.953 | 1.283 | 66.124 |
| 19 | C13 | 1.539 | 5.244 | 62.043 | 44 | H16 | 4.804 | 6.084 | 62.557 |
| 20 | C14 | 1.346 | 5.980 | 60.845 | 45 | H17 | 2.951 | 4.744 | 63.601 |
| 21 | N3 | 2.350 | 6.675 | 60.298 | 46 | H18 | 0.182 | 5.809 | 59.077 |
| 22 | C15 | 3.541 | 6.691 | 60.909 | 47 | H19 | -0.626 | 5.245 | 60.559 |
| 23 | C16 | 3.819 | 6.028 | 62.094 | 48 | H20 | -1.681 | 7.282 | 59.691 |
| 24 | C17 | 2.786 | 5.286 | 62.670 | 49 | H21 | -0.128 | 8.144 | 59.797 |
| 25 | C18 | 0.009 | 6.021 | 60.132 | 50 | H22 | -0.935 | 7.580 | 61.279 |
TABLE 5 molecular coordinates of the ryanodine receptor inhibitor c3 after geometric optimization
Table 6 coordinates in molecular dynamics simulation of the ryanodine receptor inhibitor c3
TABLE 7 molecular coordinates of the ryanodine receptor inhibitor c4 after geometric optimization
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N | -0.645 | -0.761 | -0.344 | 24 | C | 2.157 | 0.316 | -0.152 |
| 2 | C | 0.306 | -1.478 | 0.318 | 25 | C | 4.582 | 1.256 | -0.306 |
| 3 | O | 0.056 | -2.481 | 0.993 | 26 | F | 4.021 | 2.391 | -0.786 |
| 4 | H | -0.352 | 0.026 | -0.907 | 27 | F | 5.536 | 0.872 | -1.193 |
| 5 | C | -2.049 | -1.046 | -0.270 | 28 | F | 5.244 | 1.596 | 0.836 |
| 6 | C | -2.875 | -0.184 | 0.469 | 29 | H | -4.895 | 0.239 | 1.098 |
| 7 | C | -4.254 | -0.423 | 0.524 | 30 | H | -5.862 | -1.710 | -0.101 |
| 8 | C | -4.795 | -1.519 | -0.148 | 31 | H | -4.395 | -3.211 | -1.415 |
| 9 | C | -3.965 | -2.366 | -0.885 | 32 | H | -2.314 | -3.743 | -2.385 |
| 10 | C | -2.583 | -2.145 | -0.969 | 33 | H | -1.046 | -3.650 | -1.150 |
| 11 | C | -1.706 | -3.056 | -1.792 | 34 | H | -1.067 | -2.486 | -2.473 |
| 12 | C | -2.277 | 0.933 | 1.294 | 35 | H | -3.28 | 1.818 | -0.989 |
| 13 | O | -1.701 | 0.67 | 2.363 | 36 | H | -4.743 | 3.852 | -1.031 |
| 14 | N | -2.417 | 2.202 | 0.852 | 37 | H | -4.066 | 4.373 | 0.522 |
| 15 | H | -2.029 | 2.901 | 1.479 | 38 | H | -5.028 | 2.884 | 0.428 |
| 16 | C | -3.017 | 2.698 | -0.398 | 39 | H | -2.424 | 3.887 | -2.112 |
| 17 | C | -4.292 | 3.498 | -0.099 | 40 | H | -1.100 | 2.936 | -1.410 |
| 18 | C | -1.988 | 3.529 | -1.175 | 41 | H | -1.675 | 4.404 | -0.593 |
| 19 | C | 1.693 | -0.955 | 0.177 | 42 | H | 3.931 | -3.42 | 0.726 |
| 20 | N | 2.800 | -1.725 | 0.422 | 43 | H | 2.282 | -3.741 | 0.115 |
| 21 | N | 3.934 | -1.036 | 0.266 | 44 | H | 2.517 | -3.279 | 1.815 |
| 22 | C | 2.882 | -3.139 | 0.797 | 45 | H | 1.580 | 1.197 | -0.386 |
| 23 | C | 3.552 | 0.200 | -0.078 |
TABLE 8 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c4
TABLE 9 molecular coordinates of the ryanodine receptor inhibitor c5 after geometric optimization
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N | 1.075 | -0.958 | 0.836 | 27 | C | 2.597 | -0.43 | 2.702 |
| 2 | C | 0.065 | -0.038 | 0.846 | 28 | C | 1.46 | -0.334 | 3.687 |
| 3 | O | 0.158 | 1.097 | 1.317 | 29 | C | 3.232 | -1.322 | -0.961 |
| 4 | H | 1.020 | -1.715 | 0.158 | 30 | O | 2.484 | -2.3 | -1.179 |
| 5 | C | -1.208 | -0.540 | 0.254 | 31 | N | 3.849 | -0.682 | -1.977 |
| 6 | N | -2.171 | 0.292 | -0.262 | 32 | H | 3.643 | -1.092 | -2.884 |
| 7 | N | -3.246 | -0.388 | -0.698 | 33 | C | 4.516 | 0.634 | -2.01 |
| 8 | C | -2.970 | -1.668 | -0.443 | 34 | C | 5.934 | 0.492 | -2.576 |
| 9 | C | -1.706 | -1.831 | 0.149 | 35 | C | 3.668 | 1.623 | -2.822 |
| 10 | C | -2.137 | 1.715 | -0.445 | 36 | H | -1.24 | -2.747 | 0.480 |
| 11 | C | -1.486 | 2.242 | -1.562 | 37 | H | -0.96 | 4.027 | -2.642 |
| 12 | C | -1.469 | 3.621 | -1.774 | 38 | H | -2.108 | 5.544 | -1.03 |
| 13 | C | -2.114 | 4.470 | -0.871 | 39 | H | -3.277 | 4.602 | 0.947 |
| 14 | C | -2.775 | 3.948 | 0.244 | 40 | H | 5.626 | -0.885 | 0.27 |
| 15 | C | -2.785 | 2.570 | 0.455 | 41 | H | 4.095 | -0.043 | 4.189 |
| 16 | Cl | -3.601 | 1.922 | 1.865 | 42 | H | 1.835 | -0.421 | 4.710 |
| 17 | C | -3.967 | -2.723 | -0.801 | 43 | H | 0.724 | -1.127 | 3.522 |
| 18 | F | -3.573 | -3.938 | -0.349 | 44 | H | 0.931 | 0.62 | 3.592 |
| 19 | F | -4.143 | -2.837 | -2.145 | 45 | H | 4.572 | 0.987 | -0.979 |
| 20 | F | -5.194 | -2.477 | -0.277 | 46 | H | 6.434 | 1.466 | -2.588 |
| 21 | C | 2.387 | -0.73 | 1.341 | 47 | H | 5.905 | 0.114 | -3.604 |
| 22 | C | 3.479 | -0.882 | 0.456 | 48 | H | 6.535 | -0.197 | -1.974 |
| 23 | C | 4.784 | -0.736 | 0.939 | 49 | H | 4.152 | 2.605 | -2.840 |
| 24 | C | 5.005 | -0.421 | 2.279 | 50 | H | 2.672 | 1.735 | -2.384 |
| 25 | C | 3.92 | -0.267 | 3.141 | 51 | H | 3.555 | 1.282 | -3.858 |
| 26 | H | 6.018 | -0.311 | 2.652 | 52 | H | -0.998 | 1.564 | -2.254 |
TABLE 10 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c5
TABLE 11 molecular coordinates of the ryanodine receptor inhibitor c6 after geometric optimization
TABLE 12 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c6
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N1 | 2.178 | 2.433 | 60.755 | 26 | H2 | -0.341 | -2.188 | 60.245 |
| 2 | C1 | 1.580 | 3.543 | 61.287 | 27 | C17 | 0.644 | 0.984 | 59.482 |
| 3 | O1 | 0.415 | 3.592 | 61.670 | 28 | Cl2 | 0.284 | 2.25 | 58.314 |
| 4 | H1 | 2.978 | 2.562 | 60.137 | 29 | C18 | 2.593 | 0.447 | 62.728 |
| 5 | C2 | 2.429 | 4.768 | 61.356 | 30 | O2 | 3.831 | 0.359 | 62.637 |
| 6 | N2 | 2.810 | 5.477 | 62.474 | 31 | N4 | 2.006 | 0.83 | 63.885 |
| 7 | N3 | 3.596 | 6.530 | 62.165 | 32 | H3 | 2.689 | 1.019 | 64.613 |
| 8 | C3 | 3.737 | 6.469 | 60.843 | 33 | C19 | 0.617 | 0.66 | 64.352 |
| 9 | C4 | 3.025 | 5.396 | 60.278 | 34 | C20 | 0.584 | -0.307 | 65.542 |
| 10 | C5 | 2.310 | 5.376 | 63.813 | 35 | C21 | 0.008 | 2.022 | 64.701 |
| 11 | C6 | 3.016 | 4.607 | 64.637 | 36 | H4 | 2.958 | 5.121 | 59.225 |
| 12 | C7 | 2.58 | 4.455 | 65.892 | 37 | H5 | 3.165 | 3.84 | 66.575 |
| 13 | C8 | 1.411 | 5.048 | 66.366 | 38 | H6 | 1.078 | 4.881 | 67.391 |
| 14 | C9 | 0.677 | 5.858 | 65.504 | 39 | H7 | -0.239 | 6.342 | 65.842 |
| 15 | C10 | 1.136 | 6.038 | 64.199 | 40 | H8 | 1.180 | -1.821 | 62.164 |
| 16 | Cl1 | 0.258 | 7.109 | 63.112 | 41 | H9 | -0.674 | -0.440 | 58.540 |
| 17 | C11 | 4.586 | 7.481 | 60.139 | 42 | H10 | 0.035 | 0.223 | 63.541 |
| 18 | F1 | 3.883 | 8.089 | 59.161 | 43 | H11 | -0.444 | -0.429 | 65.883 |
| 19 | F2 | 5.676 | 6.921 | 59.569 | 44 | H12 | 1.190 | 0.094 | 66.354 |
| 20 | F3 | 5.034 | 8.438 | 60.981 | 45 | H13 | 0.982 | -1.274 | 65.236 |
| 21 | C12 | 1.532 | 1.185 | 60.559 | 46 | H14 | -1.017 | 1.885 | 65.044 |
| 22 | C13 | 1.737 | 0.169 | 61.52 | 47 | H15 | 0.012 | 2.66 | 63.817 |
| 23 | C14 | 1.049 | -1.043 | 61.412 | 48 | H16 | 0.595 | 2.492 | 65.49 |
| 24 | C15 | 0.196 | -1.243 | 60.333 | 49 | H17 | 3.927 | 4.113 | 64.298 |
| 25 | C16 | -0.004 | -0.252 | 59.379 |
TABLE 13 molecular coordinates of the ryanodine receptor inhibitor c7 after geometric optimization
TABLE 14 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c7
TABLE 15 molecular coordinates of the ryanodine receptor inhibitor c8 after geometric optimization
| Tag | Symbol | X | Y | Z | Tag | Symbol | X | Y | Z |
| 1 | N | -0.75 | -1.023 | -0.436 | 27 | C | -2.464 | -0.720 | -2.181 |
| 2 | C | 0.262 | -0.141 | -0.687 | 28 | C | -1.444 | -0.789 | -3.289 |
| 3 | O | 0.128 | 0.923 | -1.294 | 29 | C | -2.702 | -1.079 | 1.618 |
| 4 | H | -0.623 | -1.69 | 0.321 | 30 | O | -1.969 | -2.053 | 1.886 |
| 5 | C | 1.592 | -0.589 | -0.190 | 31 | N | -3.173 | -0.267 | 2.586 |
| 6 | N | 2.612 | 0.292 | 0.072 | 32 | H | -2.877 | -0.550 | 3.516 |
| 7 | N | 3.741 | -0.331 | 0.446 | 33 | C | -3.796 | 1.068 | 2.491 |
| 8 | C | 3.443 | -1.630 | 0.403 | 34 | C | -5.138 | 1.076 | 3.231 |
| 9 | C | 2.11 | -1.859 | 0.016 | 35 | C | -2.830 | 2.130 | 3.035 |
| 10 | C | 2.569 | 1.727 | 0.110 | 36 | H | 1.613 | -2.805 | -0.129 |
| 11 | N | 1.919 | 2.26 | 1.139 | 37 | H | 1.328 | 3.996 | 2.086 |
| 12 | C | 1.873 | 3.595 | 1.237 | 38 | H | 2.439 | 5.518 | 0.434 |
| 13 | C | 2.492 | 4.442 | 0.316 | 39 | H | 3.665 | 4.497 | -1.503 |
| 14 | C | 3.176 | 3.878 | -0.760 | 40 | H | -5.214 | -0.754 | 0.603 |
| 15 | C | 3.213 | 2.488 | -0.871 | 41 | H | -4.119 | -0.507 | -3.541 |
| 16 | Cl | 4.020 | 1.741 | -2.227 | 42 | H | -1.930 | -1.009 | -4.242 |
| 17 | C | 4.480 | -2.642 | 0.778 | 43 | H | -0.702 | -1.568 | -3.091 |
| 18 | F | 4.122 | -3.88 | 0.362 | 44 | H | -0.902 | 0.157 | -3.384 |
| 19 | F | 4.666 | -2.718 | 2.124 | 45 | H | -3.967 | 1.265 | 1.431 |
| 20 | F | 5.692 | -2.366 | 0.240 | 46 | H | -5.606 | 2.062 | 3.150 |
| 21 | C | -2.106 | -0.833 | -0.822 | 47 | H | -4.996 | 0.855 | 4.295 |
| 22 | C | -3.096 | -0.833 | 0.184 | 48 | H | -5.826 | 0.333 | 2.816 |
| 23 | C | -4.446 | -0.724 | -0.161 | 49 | H | -3.279 | 3.125 | 2.945 |
| 24 | C | -4.791 | -0.593 | -1.503 | 50 | H | -1.888 | 2.123 | 2.479 |
| 25 | C | -3.822 | -0.587 | -2.501 | 51 | H | -2.61 | 1.953 | 4.094 |
| 26 | Cl | -6.491 | -0.455 | -1.945 |
TABLE 16 coordinates in the molecular dynamics simulation of the ryanodine receptor inhibitor c8
TABLE 17 structural Table of ryanodine receptor inhibitors c1-c8 for free energy analysis
aBased on superposition of 50% of the stereoscopic field and 50% of the electrostatic fieldFull atom RMSD
1.2.2 pharmacophores of ryanodine receptor inhibitors
The ryanodine receptor is a calcium channel protein, distributed in mammals, insects, and other species. They primarily regulate the concentration of cytosolic calcium ions. An imbalance in the cytosolic calcium ion concentration when the ryanodine receptor is inhibited by the ligand results in the failure of the muscle to contract and relax normally, and the most widely used ryanodine receptor inhibitor is chlorantraniliprole. Some bisamide compounds (c1-c8) are capable of malfunctioning the ryanodine receptor (Lahm et al, 2005). c1-c8 are considered to be hot spot rings acting on RyR1, as in fig. 17a (Amador et al, 2009). This region is called a hot spot loop because mutations in amino acid residues in the loop can lead to disease. To explore the pharmacophore of bisamide ryanodine receptor inhibitors, the present examples use a variety of molecular modeling techniques to mimic the interaction of compound c8 and RyR 1. As shown in FIG. 17a, the positions of RyR1, HS-loop, and c 8; FIG. 17b shows the position of RyR1, HS-loop, and c8 in a 20ns molecular dynamics simulation; FIG. 17C shows NBO charge in effect of pyrazole/π and C-H/π; FIG. 17d shows hydrogen bonding interactions between c8 and amino acid residues on HS-loop; FIG. 17e shows a pharmacophore c 8.
1.2.3 free energy calculation of ligand-receptor complexes
Compound c8 was docked to the hotspot ring using AutoDock software. As shown in fig. 18b, the rms deviation is plotted against the energy-lowest constellation. The c8-RyR1 complex is subjected to 2ns molecular dynamics simulation, and the root mean square deviation of proteins and small molecules in the simulation process is less than
(FIG. 18b 2). The invention then extends the simulation to 20ns, and it was found that the rms deviation within 0 to 75ns varies less than the same
(FIG. 18b 1). As shown in FIG. 18a, during the 20ns simulation, Energy (ETOT), potential Energy (EPTOT), Temperature (TEMP) and kinetic Energy (EKTOT) are atIn an equilibrium state.
The present examples analyzed the contribution of amino acid residues R157, R164 and D167 to the reduction of binding free energy using an alanine scan. These amino acid residues were selected by the present examples because they were shown to be closely related to disease (Amador et al, 2009). As shown in Table 3.18, the mutation of these three amino acid residues results in a decrease of-2 kcal/mol in the free energy of ligand-receptor binding, indicating that they are important for the binding of the inhibitor to the receptor.
The inventive examples used the same method to evaluate the interaction of seven other bisamide compounds (c1-c7) with the receptor. Mutations in R157, R164 and D167 resulted in a significant reduction in binding free energy (table 18). The relative positions of these three amino acid residues were not changed in a kinetic simulation of 20ns, as shown in FIG. 17 b. Calculations demonstrate that these three amino acid residues play a crucial role in inhibitor-receptor interactions. The present examples use quantum mechanical methods to analyze the details of the interaction of c8 and these three amino acid residues in order to discover the pharmacophore of RyR inhibitors.
TABLE 18 alanine scan results (kcal/mol) for compounds c1-c8 and R157, R164 and D167a
aThe calculation is carried out on the basis of 2 nanosecond molecular dynamics
bA homology MODEL for XP _002080659(4-199) was automatically generated using SWISS-MODEL, and the template used 3 HSM-A. The root mean square deviation of all atoms of HS-loops between them is only
The sequence identity between these two proteins was 61%
cA threshold for the change in calcium ion concentration (. mu.M) is cited from (Lahm et al, 2005)
dSum of alanine scan values
1.3 Quantum mechanics approach to study the interaction of c8 with ryanodine receptor
The present invention uses the energy-lowest conformation of the c8-RyR1 complex in a 20 nanosecond kinetic simulation for single point analysis. NBO charges for single-point pyrazole/π interaction and C-H/π interaction were 0.041(115C), -0.832(45N) and 0.295(65H), respectively (FIG. 17C). The NBO charge indicates that they have a wide range of interactions. The present invention hereby assumes that the pyrazole ring on c8 plays an important role in the interaction between molecules.
The most compelling evidence for hydrogen bonding is the calculation of laplace values and charge densities at the saddle points of the bonds. S154, Q156, R157, R164, G166, D167 and D168 form 2, 5, 1, 3 and 2 hydrogen bonds with the ligand molecule, respectively (table 5 and fig. 17D). The charge densities of R157-1(0.03175a.u.), D167-2(0.03660a.u.), and R157-5(0.01886a.u.) were higher than those of the other hydrogen bonds (Table 19), indicating that the hydrogen bonds formed by N108-H111, O137 and N118 on c8 and R157, D167 were important (FIG. 17 e).
The present invention also obtains a stabilized free energy Δ E (2) by natural bond orbital analysis. The free energy of stabilization for R157-1, R157-5 and D167-2 was significantly higher than for the other bonds (Table 20 and FIG. 19), this calculation and the Δ Δ G of the inventionbindR157 and Δ Δ GbindThe D167 results and the charge density analysis results were consistent.
The present invention confirmed that N108-H111, O137, the pyrazole ring and N118 are pharmacophores of c8 by atomic and natural bond orbital analysis in the molecule.
TABLE 19 charge density and Laplace values for c8 and major amino acid residues at B3LYP/6-31+ G (. lambda.) levels
TABLE 20 c8 and stabilization free energy (kcal/mol) of major amino acid residues
aCalculation at B3LYP/6-31+ G (. multidot.) levels
The inventive example simulates the interaction of bisamides and ryanodine receptors using a force field-based approach and finds that c8 forms stable pi-pi interactions with key amino acid residues on HS-loop. On the basis, the charge density of a saddle point formed by c8 and atoms on surrounding amino acids, a Laplace operator and stabilizing free energy are calculated by using a quantum mechanical method. The results of calculations obtained by various methods indicate that the pharmacophores of c8 interacting with ryanodine receptor are N108-H111, O137, pyrazole ring and N118. In the embodiment of the invention, 11 compounds are designed and synthesized by pharmacophore analysis. As described below.
Synthesis of Compounds d1-d11
2.1 Synthesis method
Compound d1 preparation procedure: 0.15g (0.5mmol) of Compound A (shown in FIG. 21) was dissolved in 10mL SOCl2And refluxed for 1 hour for excess SOCl2Removing by vacuum pumping, dissolving the residue in 5mL pyridine, adding 0.5mmol methyl 2-aminothiophene-3-carboxylate (methyl 2-aminothiophene-3-carboxylate) dissolved in pyridine, concentrating the mixture under reduced pressure after 1 hour, extracting the residue with 5mL water and 20mL ethyl acetate, washing the organic phase with 5mL brine, drying with anhydrous sodium sulfate, evaporating the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound d1 shown in formula II with the yield of 90%;
compound d2 preparation procedure: dissolving 0.22g (0.5mmol) of compound d1 in 20mL of chloroform, adding 0.6mmol of nitrogen bromosuccinimide dissolved in chloroform, distilling the mixture under reduced pressure after 10 hours, and performing silica gel column chromatography on the residue to obtain a compound d2 shown in formula III with the yield of 83%;
preparation steps of compounds d3-d 11: dissolving 0.5mmol of compound d2 in 2mL of a mixture of triethanolamine and dimethylformamide in a volume ratio of 1: 1; the corresponding alkyne derivative (0.5mmol), cuprous iodide (10mg,0.05 mmol) and PdCl were then added2(PPh3)2(35mg,0.05mmol) was added to the solution and stirred at 85 deg.C for 12 hours, thenThe mixture was cooled to room temperature and filtered over celite, the celite was washed with ethyl acetate, the washings were concentrated under reduced pressure, the residue was extracted with 3ml of water and 10ml of ethyl acetate, the organic phase was washed with 5ml of brine, dried over anhydrous sodium sulfate, the organic solvent was removed in vacuo, and the mixture was purified by silica gel column chromatography to give one of the compounds d3-d11 of formulae v to XIII in 33-45% yield (fig. 21).
In addition, the inventive example yielded a single crystal structure of compound d8 to clarify the conformation of the synthesized molecule (FIG. 22). The rigid coplanar structure of compound d8 forms a large conjugated system and this favors the excited state generation of the molecule.
Figure 20 shows a scheme for designing new photosensitizers based on the pharmacophores of b1 and c8, when the corresponding alkyne derivative is compound b2, the resulting product is compound d3 (formula v). Correspondingly, when the corresponding alkyne derivative is the compound b3, the product prepared is the compound d4 (formula VI); when the corresponding alkyne derivative is compound b4, the product obtained is compound d5 (formula VII); when the corresponding alkyne derivative is compound b5, the prepared product is compound d6 (formula VIII); when the corresponding alkyne derivative is compound b6, the product obtained is compound d7 (formula ix); when the corresponding alkyne derivative is compound b7, the product prepared is compound d8 (formula X); when the corresponding alkyne derivative is compound b8, the product prepared is compound d9 (formula XI); when the corresponding alkyne derivative is compound b9, the product obtained is compound d10 (formula XII); when the corresponding alkyne derivative is compound b10, the product prepared is compound d11 (formula XIII).
The structural formulas of the compound A and the compound b 1-10 are shown as follows:
the structural formulas, physicochemical properties and spectral information of the compounds d1-d11 are respectively as follows:
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) thiophane-3-carboxylate (formula II, compound d 1.) as a white solid in 90% yield, m.p. 193.2-194.0 ℃,1H-NMR(CDCl3,600MHz):＝11.85(s,1H),8.52(d,J＝4.8Hz,1H),7.94(d,J＝7.8Hz,1H),7.46(dd,J1＝8.4Hz,J2＝4.2Hz,1H),7.24(d,J＝5.4Hz,1H),7.06(s,1H),6.78(d,J＝5.4Hz,1H),3.95(s,3H).13C-NMR(CDCl3,150MHz):166.2,153.6,148.5,147.6,147.0,139.2,137.7,129.1,128.3,126.0,123.9,116.9,113.7,110.6,52.0.MS(ESI-)m/z:441.3(M-1).Anal.Calcd.for C15H10BrClN4O3S:C 40.79,H 2.28,N 12.68；Found:C 40.85,H 2.16,N 12.74.
methyl5-bromo-2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) thiophene-3-carboxylate (formula III, compound d2) as a white solid, yield 83%, m.p. 229.4-230.9 ℃,1H-NMR(CDCl3,600MHz):＝11.81(s,1H),8.50(d,J＝4.8Hz,1H),7.93(d,J＝8.4Hz,1H),7.45(dd,J1＝7.8Hz,J2＝4.8Hz,1H),7.21(s,1H),7.03(s,1H),3.92(s,3H).13C-NMR(CDCl3,150MHz):165.2,153.7,147.8,147.1,139.3,130.9,129.1,128.8,128.3,126.1,125.8,113.6,110.8,105.3,52.2.MS(ESI-)m/z:519.2(M-1).Anal.Calcd.for C15H9Br2ClN4O3S:C 34.61,H 1.74,N 10.76；Found:C 34.73,H1.56,N 10.84.
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) -5- (phenylethynyl) thiophene-3-carboxylate (formula V, compound d3) as a yellow solid, 43% yield, m.p. 217.3-219.0 ℃,1H-NMR(CDCl3,600MHz):＝11.95(s,1H),8.54(d,J＝4.2Hz,1H),7.96(d,J＝9.0Hz,1H),7.59(t,2H),7.46(d,J＝7.2Hz,2H),7.41(s,1H),7.39(d,J＝4.8Hz,1H),7.34(d,J＝7.8Hz,1H),7.24(s,1H),3.95(s,3H).13C-NMR(CDCl3,150MHz):165.7,154.3,148.9,147.8,147.0,139.2,137.0,136.0,131.8(2C),128.9,128.4(2C),128.2,126.0,122.6,122.1,114.7,113.3,111.3,93.4,81.7,52.2.MS(ESI-)m/z:563.5(M-1+Na).Anal.Calcd.for C23H14BrClN4O3S:C 50.99,H 2.60,N 10.34；Found:C 50.85,H 2.51,N 10.44.
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) -5- (p-tolythinyl) thiophene-3-carboxylate (formula VI, compound d4) as a yellow solid, 38% yield, mp 196.4-197.0 deg.C,1H-NMR(CDCl3,600MHz):＝11.89(s,1H),8.52(d,J＝4.8Hz,1H),7.93(d,J＝8.4Hz,1H),7.46(dd,J1＝8.4Hz,J2＝4.8Hz,1H),7.37(s,1H),7.36(d,J＝8.4Hz,2H),7.15(d,J＝8.4Hz,2H),7.06(s,1H),3.95(s,3H),2.36(s,3H).13C-NMR(CDCl3,150MHz):165.8,153.7,148.5,147.1,145.1,139.3,137.4,131.9,131.3(2C),129.1,128.3,127.9(2C),127.8,126.1,119.7,115.2,113.5,110.8,93.8,81.0,52.2,35.3.MS(ESI-)m/z:555.3(M-1).Anal.Calcd.for C24H16BrClN4O3S:C 51.86,H 2.90,N 10.08；Found:C 51.95,H 2.81,N 10.10.
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamid) -5- ((4-ethylphenyl) ethyl) thiophene-3-carboxylate (formula VII, Compound d 5.) as a yellow solid, yield: 45%, m.p. 196.2-198.1 ℃,1H-NMR(CDCl3,600MHz):＝11.89(s,1H),8.52(d,J＝4.8Hz,1H),7.94(d,J＝8.4Hz,1H),7.47(dd,J1＝7.8Hz,J2＝4.2Hz,1H),7.38(s,1H),7.37(d,J＝8.4Hz,2H),7.15(d,J＝8.4Hz,2H),7.06(s,1H),3.95(s,3H),2.66(q,2H),1.24(s,3H).13C-NMR(CDCl3,150MHz):165.7,153.7,148.5,147.0,145.1,139.2,137.4,131.8,131.4(2C),129.1,128.3,127.9(2C),127.8,126.1,119.7,115.2,113.5,110.7,93.8,80.9,52.2,28.8,15.3.MS(ESI-)m/z:569.4(M-1).Anal.Calcd.for C25H18BrClN4O3S:C 52.69,H 3.18,N 9.83；Found:C 52.74,H 3.11,N 9.90.
methyl2-(3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-caroxamido) -5- ((4-propylphenyl) ethyl) thiophene-3-carboxylate (formula VIII, compound d 6.) as a yellow solid, yield: 37%, m.p. 200.1-201.3 deg.C,1H-NMR(CDCl3,600MHz):＝11.89(s,1H),8.52(d,J＝4.8Hz,1H),7.94(d,J＝7.8Hz,1H),7.47(dd,J1＝8.4Hz,J2＝4.8Hz,1H),7.38(s,1H),7.36(d,J＝9.0Hz,2H),7.15(d,J＝8.4Hz,2H),7.07(s,1H),3.95(s,3H),2.60(t,J＝7.8Hz,2H),1.63-1.68(m,2H),0.95(t,J＝7.8Hz,3H).13C-NMR(CDCl3,150MHz):165.8,153.7,148.5,147.4,147.0,143.6,139.3,137.4,131.7,131.3(2C),128.5(2C),128.3,127.9,126.0,119.7,115.2,113.5,110.7,93.8,81.0,52.2,37.9,24.3,13.7.MS(ESI-)m/z:583.4(M-1).Anal.Calcd.for C26H20BrClN4O3S:C 53.48,H 3.45,N 9.60；Found:C 53.59,H 3.31,N 9.71.
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) -5- ((4-butylphenyl) ethyl) thiophene-3-carboxylate (formula IX, compound d7) as a yellow solid, 43% yield, m.p. 200.2-201.5 ℃,
1H-NMR(CDCl3,600MHz):＝11.86(s,1H),8.52(d,J＝4.8Hz,1H),7.94(d,J＝8.4Hz,1H),7.45(dd,J1＝8.4Hz,J2＝4.8Hz,1H),7.37(s,1H),7.36(d,J＝9.0Hz,2H),7.16(d,J＝7.8Hz,2H),7.06(s,1H),3.96(s,3H),2.63(t,J＝7.2Hz,2H),1.59-1.64(m,2H),1.34-1.40(m,2H),0.95(t,J＝7.2Hz,3H).13C-NMR(CDCl3,150MHz):165.7,153.6,148.5,147.0,139.2,137.4,132.3,131.3(2C),130.9,128.8,128.3,127.9(2C),127.6,126.1,115.4,114.6,113.4,110.7,93.6,81.1,52.2,35.7,33.3,22.3,13.9.MS(ESI-)m/z:597.3(M-1).Anal.Calcd.for C27H22BrClN4O3S:C54.24,H 3.71,N 8.03；Found:C 54.34,H 3.62,N 8.10.
methyl2- (3-bromo-1- (3-chloropyridin-2-yl) -1H-pyrazole-5-carboxamido) -5- ((4-methoxyphenyl) ethyl) thiophene-3-carboxylate (formula X, compound d8) as a yellow solid, 42% yield, m.p. 200.3-201.5 ℃,1H-NMR(CDCl3,600MHz):＝11.87(s,1H),8.54(d,J＝4.2Hz,1H),7.97(d,J＝8.4Hz,1H),7.48(dd,J1＝8.8Hz,J2＝4.8Hz,1H),7.43(d,J＝9.0Hz,2H),7.38(s,1H),7.09(s,1H),6.91(d,J＝8.4Hz,2H),3.99(s,3H),3.88(s,3H).13C-NMR(CDCl3,150MHz):167.7,165.7,159.1,153.6,148.5,147.2,147.0,139.2,137.5,132.3,130.92,129.12,128.8,128.3,127.6,126.1,115.3,114.6,113.5,110.7,93.6,80.3,55.3,52.2.MS(ESI-)m/z:571.4(M-1).Anal.Calcd.for C24H16BrClN4O4S:C 50.41,H 2.82,N 9.80；Found:C 50.55,H 2.71,N 9.71.
methyl2- (3-bromo-1- (3-fluoropyridin-2-yl) -1H-pyrazole-5-carboxamid) -5- ((4- (trifluoromethyl) phenyl) ethyl) thiophene-3-carboxalate (formula XI, compound d 9.) as a yellow solid, yield 35%, m.p. 190.7-192.2 ℃,1H-NMR(CDCl3,600MHz):＝11.92(s,1H),8.53(d,J＝4.8Hz,1H),7.94(d,J＝7.8Hz,1H),7.47(dd,J1＝7.8Hz,J2＝4.2Hz,1H),7.44(d,J＝9.0Hz,2H),7.39(s,1H),7.06(s,1H),7.04(t,J＝8.4Hz,2H),3.95(s,3H).13C-NMR(CDCl3,150MHz):166.2,165.6,153.7,153.6,148.5,148.4,148.0,147.0,139.2,137.4,128.8,128.3,127.7,126.1,126.0,123.9,115.3,114.6,113.4,110.8,110.7,92.1,84.2,52.2.MS(ESI-)m/z:609.3(M-1).Anal.Calcd.for C24H13BrClF3N4O3S:C 47.27,H 2.15,N 9.19；Found:C 47.20,H 2.01,N 9.28.
methyl2- (3-bromo-1- (3-fluoropyridin-2-yl) -1H-pyrazole-5-carboxamid) -5- ((4-fluorophenyl) ethyl) thiophene-3-carboxalate (formula XII, Compound d10) as a yellow solid, 43% yield, m.p. 218.0-219.4 ℃,1H-NMR(CDCl3,600MHz):＝11.90(s,1H),8.52(d,J＝4.8Hz,1H),7.93(d,J＝8.4Hz,1H),7.46(dd,J 1＝7.8Hz,J2＝4.2Hz,1H),7.44(d,J＝5.4Hz,1H),7.43(d,J＝5.4Hz,1H),7.39(s,1H),7.06(s,1H),7.04(t,J＝8.4Hz,2H),3.95(s,3H).13C-NMR(CDCl3,150MHz):165.7,163.5,161.8,153.7,148.4,147.5,147.0,139.2,137.3,133.3,133.2,129.2,128.3(2C),126.1,118.6(2C),115.7,113.5,110.8,92.4,81.3,52.2.MS(ESI-)m/z:559.3(M-1).Anal.Calcd.for C23H13BrClFN4O3S:C 49.35,H 2.34,N 10.01；Found:C 49.45,H 2.21,N 10.04.
methyl2- (3-bromo-1- (3-fluoropyridin-2-yl) -1H-pyrazole-5-carboxamid) -5- ((4-nitrophenyl) ethyl) thiophene-3-carboxalate (formula XIII, Compound d11) as a yellow solid, 33% yield, m.p. 211.2-212.1 ℃,1H-NMR(CDCl3,600MHz):＝11.85(s,1H),8.52(d,J＝4.8Hz,1H),8.21(d,J＝9.0Hz,1H),7.95(dd,J1＝9.0Hz,J2＝4.2Hz,1H),7.51(s,1H),7.45-7.48(m,2H),7.24(d,J＝6.0Hz,1H),7.07(s,1H),6.78(d,J＝6.0Hz,1H),3.96(s,3H).13C-NMR(CDCl3,150MHz):166.3,165.7,153.7,153.6,148.5,147.2,147.0,139.2,137.4,132.3,130.9,128.8,128.3,127.6,126.1,126.0,115.3,114.6,113.4,110.7,91.8,87.2,52.2.MS(ESI-)m/z:586.3(M-1).Anal.Calcd.for C23H13BrClN5O5S:C 47.08,H 2.23,N 11.93；Found:C 47.20,H 2.11,N 11.98.
third, evaluation of Effect
The novel structure provided by the embodiment of the invention has good biological activity on Staphylococcus aureus (Staphylococcus aureus) and influenza virus.
3.1 biological Activity assay of Compounds
Activity test method (turbidimetry) against Staphylococcus aureus (26076): preparing hydrolyzed casein (Mueller-Hinton) broth culture medium (MH broth culture medium), subpackaging in test tubes, each 9mL, sterilizing, adding medicinal liquid to prepare a series of concentration gradients, inoculating the same amount of bacterial liquid, inoculating the same bacterial liquid with the non-medicinal MH broth culture medium as a control, culturing in a constant temperature incubator at 37 deg.C for 2h, dividing into two groups, and placing one group under UV-A ultraviolet lamp (illumination intensity of 2074 μ W/cm)2) Irradiating for 1 hr, culturing the other group in dark place, after the irradiation treatment, placing all the treatments in a constant temperature incubator, culturing in dark place for 15 hr, examining the result, measuring absorbance of each treatment solution at 480nm with 721 type spectrophotometer, calculating growth inhibition rate, and calculating the concentration value (IC) of the inhibition medium50)。
Experiments prove that under the condition of illumination, the bactericidal activity of the compound provided by the invention is greatly enhanced, and the activity to staphylococcus aureus (26076) is shown in table 21.
TABLE 21 IC of active Compounds on Staphylococcus aureus50Value (15h)
3.2 biological Activity assay of Compounds
Activity assay method for influenza virus mouse lung adapted strain FM1 (immunofluorescence method): monolayer-grown Hep-1 cell plates were infected with FM1(100 TCID)50) Put at 37 ℃ CO2Adsorbing for 1 hr in constant temperature incubator, washing with 0.01mol/L PBS buffer solution (pH7.4), adding maintenance solution containing different compounds (to final concentration of 100 μ g/mL in culture system), culturing for 3 hr in dark place, dividing into 2 groups, and placing one group under UV-A ultraviolet lamp (illumination intensity of 2074 μ W/cm)2) And (4) illuminating for 1h, culturing the other group in a dark place, and continuously culturing all the groups in the dark place after the illumination treatment of the illumination group is finished. After 10h of adsorption, the 4-well drug-containing maintenance solution was aspirated, washed with PBS 2 times, and fixed with 95% ethanol for 10 min. During staining, corresponding rabbit anti-FM 1 immune serum is added firstly, the mixture is cultured for 2h at 37 ℃, taken out and washed by PBS, anti-goat anti-rabbit IgG-FITC labeled antibody is cultured and rinsed by the same method, and finally the amount of specific fluorescent cells is observed by an epifluorescence microscope. The ratio of the total cell surface is divided into 5 grades (0 grade: 0%; 1 grade: less than 5%; 2 grade: 5% -10%; 3 grade: 10% -30%; 4 grade: more than 30%). Experiments prove that the compound provided by the invention has an inhibitory effect on viruses under the condition of illumination, and the activity on influenza virus mouse lung adaptive strain FM1 is shown in Table 22.
TABLE 22 Effect of the active Compounds on the intracellular proliferation of influenza Virus mouse lung Adaptation strain FM1(10 h)
| Active ingredient | Light-resistant group | Illumination group |
| Compound d3 | 2332 | 0000 |
| Compound d4 | 2332 | 0000 |
| Compound d5 | 3232 | 0000 |
| Compound d6 | 3223 | 0000 |
| Compound d7 | 2332 | 0000 |
| Compound d8 | 3223 | 0000 |
| Compound d9 | 2332 | 0000 |
| Compound d10 | 2332 | 0000 |
| Compound d11 | 3232 | 0000 |
| Control | 4444 | 4444 |
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. A novel organic compound having the structure of formula I:
in the formula I, R1Is H or halogen.
2. The novel organic compound of claim 1, having the structure of formula ii or formula iii:
3. a method for preparing the novel organic compound according to claim 2, wherein when the structure of the novel organic compound is represented by formula ii, the method comprises:
compound d1 preparation procedure: dissolving Compound A in SOCl2And refluxing the excess SOCl2Vacuum-pumping off, dissolving the remainder in pyridine, and adding pyridinePyridine methyl 2-aminothiophene-3-carboxylate, concentrating the mixture under reduced pressure, extracting the residue with water and ethyl acetate, washing the organic phase with brine, drying with anhydrous sodium sulfate, evaporating to remove the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound shown as a formula II, which is recorded as a compound d 1;
when the structure of the novel organic compound is shown as a formula III, the preparation method comprises the following steps:
compound d1 preparation procedure: dissolving Compound A in SOCl2And refluxing the excess SOCl2Vacuum pumping to remove residues, dissolving the residues in pyridine, adding methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, then concentrating the mixture under reduced pressure, extracting the residues with water and ethyl acetate, washing an organic phase with brine, drying the organic phase with anhydrous sodium sulfate, evaporating to remove an organic solvent after drying, and purifying the residues by using a silica gel column chromatography to obtain a compound d1 shown in formula II;
compound d2 preparation procedure: dissolving a compound d1 in chloroform, adding nitrogen bromosuccinimide dissolved in chloroform, then distilling the mixture under reduced pressure, and carrying out silica gel column chromatography on the residue to obtain a compound shown as a formula III, wherein the compound is marked as a compound d 2;
the structural formula of the compound A is shown as follows:
4. the method for producing the novel organic compound according to claim 3, wherein in the step of producing the compound d1, 0.15g of the compound A is dissolved in 10mL of SOCl2And refluxed for 1 hour for excess SOCl2Removing by vacuum pumping, dissolving the residue in 5mL of pyridine, adding 0.5mmol of methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, concentrating the mixture under reduced pressure after 1 hour, extracting the residue with 5mL of water and 20mL of ethyl acetate, washing an organic phase with 5mL of brine, drying with anhydrous sodium sulfate, evaporating to remove the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound d1 shown in the formula II;
in the preparation step of the compound d2, 0.22g of the compound d1 is dissolved in 20mL of chloroform, 0.6mmol of nitrogen bromosuccinimide dissolved in chloroform is added, after 10 hours, the mixture is distilled under reduced pressure, and the residue is subjected to silica gel column chromatography to obtain the compound d2 shown in the formula III.
5. A pharmaceutical composition comprising the novel organic compound of claim 1 and having the structure of formula iv:
in the formula IV, R2Is H, alkyl, alkoxy, -CF3One of halogen or nitro.
6. The drug of claim 5, having the structure of formulae V to XIII, as compounds d3 to d 11:
7. a process for the preparation of a medicament according to claim 6, wherein compounds d3-d11 are prepared by a process comprising the steps of:
compound d1 preparation procedure: dissolving Compound A in SOCl2And refluxing the excess SOCl2Vacuum pumping to remove residues, dissolving the residues in pyridine, adding methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, then concentrating the mixture under reduced pressure, extracting the residues with water and ethyl acetate, washing an organic phase with brine, drying the organic phase with anhydrous sodium sulfate, evaporating to remove an organic solvent after drying, and purifying the residues by using a silica gel column chromatography to obtain a compound d1 shown in formula II;
compound d2 preparation procedure: dissolving a compound d1 in chloroform, adding nitrogen bromosuccinimide dissolved in chloroform, then distilling the mixture under reduced pressure, and carrying out silica gel column chromatography on the residue to obtain a compound d2 shown in a formula III;
preparation steps of compounds d3-d 11: dissolving the compound d2 in a mixture of triethanolamine and dimethylformamide, and then adding the predetermined alkyne derivative, cuprous iodide and PdCl2(PPh3)2Adding the mixture into the solution, heating and stirring, cooling the mixture to room temperature, filtering the mixture by using kieselguhr, washing the kieselguhr by using ethyl acetate, concentrating the washing liquor under reduced pressure, extracting the residue by using water and ethyl acetate, washing an organic phase by using brine, drying the organic phase by using anhydrous sodium sulfate, vacuumizing the organic solvent, and purifying the mixture by using a silica gel column chromatography to obtain one of compounds d3-d11 shown in formulas V-XIII;
the structural formula of the compound A is shown as follows:
8. the process for the preparation of a medicament according to claim 7, wherein the process for the preparation of compounds d3-d11 comprises the following steps:
compound d1 preparation procedure: 0.15g of Compound A was dissolved in 10mL of SOCl2And refluxed for 1 hour for excess SOCl2Removing by vacuum pumping, dissolving the residue in 5mL of pyridine, adding 0.5mmol of methyl 2-aminothiophene-3-carboxylate dissolved in pyridine, concentrating the mixture under reduced pressure after 1 hour, extracting the residue with 5mL of water and 20mL of ethyl acetate, washing an organic phase with 5mL of brine, drying with anhydrous sodium sulfate, evaporating to remove the organic solvent after drying, and purifying the residue by silica gel column chromatography to obtain a compound d1 shown in the formula II;
compound d2 preparation procedure: dissolving 0.22g of compound d1 in 20mL of chloroform, adding 0.6mmol of nitrogen bromosuccinimide dissolved in chloroform, distilling the mixture under reduced pressure after 10 hours, and performing silica gel column chromatography on the residue to obtain a compound d2 shown in the formula III;
preparation steps of compounds d3-d 11: 0.5mmol of compound d2 was dissolved in 2mL of a mixture of triethanolamine and dimethylformamideIn the product, triethanolamine and dimethylformamide are mixed according to the volume ratio of 1: 1; 0.5mmol of the predetermined alkyne derivative, 0.05mmol of cuprous iodide and 0.05mmol of PdCl2(PPh3)2Adding the mixture into the solution, stirring at 85 ℃ for 12 hours, cooling the mixture to room temperature, filtering the mixture by using kieselguhr, washing the kieselguhr by using ethyl acetate, concentrating the washing solution under reduced pressure, extracting the residue by using 3ml of water and 10ml of ethyl acetate, washing an organic phase by using 5ml of saline, drying the organic phase by using anhydrous sodium sulfate, vacuumizing an organic solvent, and purifying the mixture by using silica gel column chromatography to obtain one of compounds d3-d11 shown in formulas V-XIII.
9. The process for the preparation of a medicament according to claim 7, wherein in the steps of preparing compounds d3-d11, when the medicament is compounds d3-d11, respectively, the predetermined alkyne derivative is compounds b2-b 10:
10. use of a medicament according to claim 7, or a pharmaceutically acceptable salt thereof, for inhibiting staphylococcus aureus and influenza virus.
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