SI24317A - New carboxamide inhibitors of the enzyme MurD - Google Patents

Abstract

The invention relates to inhibitors of the UDP-N-acetylmuramyl L-Ala: D-Glu ligase (MurD) enzyme inhibitor and their use for the development of active substances for the treatment of bacterial infections by the inhibition of MurD ligase.

Description

The invention relates to the field of pharmaceutical chemistry and relates to inhibitors of the enzyme UDP- / Vacetylmuramyl L-Ala: D-Glu ligase (MurD), which are potentially useful for the development of antibacterial agents.

A technical problem

The increasing resistance of bacteria to antibacterial agents is a very serious problem. Many infectious diseases are at risk of becoming unmanageable. The development of new antibacterial agents is one of the necessary measures in the fight against bacterial resistance. When developing new antimicrobial agents, there is a need to identify new targets and related new antibacterial agents.

The state of the art

The MurD enzyme is a bacterial protein involved in the intracellular stages of bacterial peptidoglycan synthesis. It is essential for bacterial growth and is therefore an interesting target for the development of antimicrobial agents (van Heijenoort, J. Nat. Prod. Rep. 2001, 18, 503-19). The enzyme catalyzes the attachment of D-glutamic acid to the UDP-MurNAc-L-Ala (UMA) substrate:

UDP-MurNAc-L-Ala + D-Glu + ATP <-> UDP-MurNAc-L-Ala-D-Glu + ADP + Pi

The structure of the MurD enzyme is known, and several crystal structures of the protein itself and the protein in complex with substrates and various inhibitors are available. We also have data on the dynamic properties of the protein obtained from NMR data and molecular dynamics simulations (Simčič, M.; Hodošček, M.; Humljan, J.; Kristan, K.; Urleb, U.; Kocjan, D. Golic Grdadolnik, SJ Med Chem Chem 2009, 52, 2899-2908, Tomasic, T .; Zidar, N .; Kovac, A .; Turk,

S .; Simcic, M.; Blanot, D.; Muller-Premru, M.; Filipic, M.; Grdadolnik, S. G.; Zega, A.; Anderluh, M.; Gobec, S.; Kikelj, D.; Peterlin Masic, L. ChemMedChem 2010, 5, 286-295, Simcic, M.; Sosic, I.; Hodošček, M.; Barreteau, H.; Blanot, D.; Gobec, S.; Grdadolnik, S. G. PLoS ONE 2012). All this provides a good basis for the structurally supported search for novel MurD enzyme inhibitors.

There are several groups of inhibitors that act on MurD ligase. The first effective inhibitors of MurD ligase were phosphinate inhibitors (Tanner, M. E.; Vaganay, S.; van Heijenoort, J.; Blanot, D. J. Org. Chem. 1996, 61, 1756-1760). Macrocyclic inhibitors are also known (Horton, JR; Bostock, JM; Chopra, I .; Hesse, L .; Phillips, SE; Adams, DJ; Johnson, AP; Fishwick, CW Bioorg. Med. Chem. Lett. 2003, 13; 1557-60), sulfonamide inhibitors (Kotnik, M.; Humljan, J.; Contreras-Martel, C.; Oblak, M.; Kristan, K.; Herve, M.; Blanot,

D .; Urleb, U.; Gobec, S.; Dessen, A.; Scholmayer, T. J. Mol. Biol. 2007, 370, 107-115, Humljan, J.; Kotnik, M.; Contreras-Martel, C.; Blanot, D.; Urleb, U.; Dessen, A.; Solmajer, T.; Gobec, S. J. Med. Chem. 2008, 51, 7486-7494), thiazolidine-2,4-dione inhibition ((Tomasic, T.; Zidar, N.; Rupnik, V.; Kovac, A.; Blanot, D.; Gobec, S.; Kikelj , D.; Mašič, LP Bioorg., Med. Chem. Lett. 2009, 19, 153-7; Tomašič, T.; Zidar, N.; Šink, R.; Kovač, A.; Blanot, D.; Contreras- Martel, C.; Dessen, A.; Muller-Premru, M.; Zega, A.; Gobec, S.; Kikelj, D.; Peterlin Masic, L. J. Med. Chem. 2011, 54, 4600-4610) and V. -acylhydrazone inhibitors.

The inhibitors listed above have too little antibacterial activity to be directly used as active ingredients for the treatment of bacterial infections. There is still a need for new, more effective MurD ligase inhibitors that would be useful as antibacterial agents. So far, the dynamic properties of the protein have not been taken into account in the design of MurD inhibitors.

The literature states that carboxamide inhibitors, which are also the subject of this patent application, have an inhibitory effect on the growth of Mycobacterium tuberculosis, but the mechanism of action is unknown (Ukrainets, IV; Mospanova, EV; Sidorenko, LV Chemistry of Heterocyclic Compounds 2007, 43). 863-870, Ukrainets, I.; Grinevich, L.; Tkach, A.; Gorokhova, O.; Kravchenko, V.; Sim, G. Chemistry of Heterocyclic Compounds 2011, 46, 1364-1370). There is no information on the inhibitory effect on the growth of other bacteria.

Detailed description of the invention with embodiments

The invention relates to compounds I and II and their pharmaceutically acceptable salts for use for inhibiting MurD ligase

N- (1H-benzoimidazol-2-yl) -2-hydroxy-1-methyl-4-oxo-quinoline-3-carboxamide

N- (1H-benzo [d] imidazol-2-yl) -6-hydroxy-4-oxo-2,4-dihydro-1H-pyrrolo [3,2,1-zy] quinoline-5carboxamide.

Compounds I and II and their pharmaceutically acceptable salts represent novel potent inhibitors of MurD ligase as evidenced by assays for determining the IC50 constant and residual activity (RA) of the MurD enzyme (ie RA 12% (at 100 μΜ compound concentration) and IC50 = 17 μΜ for Compound I and RA 10% (at 25 μΜ compound concentration) and IC50 = 5 μΜ for compound II).

The invention also relates to compounds I and II and their pharmaceutically acceptable salts for use in the treatment of bacterial infections. Said treatment of bacterial infections is preferably by inhibition of MurD ligase.

The invention further provides pharmaceutical forms containing compound I and / or compound II and / or pharmaceutically acceptable salts thereof for use in the treatment of bacterial infections, or pharmaceutical forms containing compound I and / or compound II and / or their pharmaceutical acceptable salts for use in inhibiting MurD ligase. Preferred treatment for bacterial infections is through inhibition of MurD ligase. The pharmaceutical forms of the present invention may be e.g. tablets, capsules, pellets, granules or combinations thereof. These may be immediate-form and modified-release pharmaceutical forms. The pharmaceutical forms of the present invention further comprise at least one other pharmaceutically acceptable excipient.

Virtual Grating

1. Principle

In virtual screening using a computer (and silico), we look for compounds that would have the desired inhibitory activity on the MurD protein. Structures are obtained from compound libraries such as the freely available ZINC database. With structurally supported virtual lattice, we know the 3d dimensional structure of the target. Several crystal structures have been reported for the MurD protein. The molecules are anchored into the selected binding site, and then their binding energy is estimated using the evaluation functions and thus the activity is inhibited.

2. Workflow

a) Selection of binding site for anchoring molecules

In selecting the binding site, we have taken into account our extensive studies of the dynamic properties of MurD in complexes with various inhibitors, such as: first generation sulfonamide inhibitors second generation sulfonamide inhibitors, hydroxy substituted 5benzelidentiazolidin-4-one and 2-thioxothiazolidin-4-one inhibitor NMR methods and molecular dynamics (MD) simulations.

Our research has shown a noticeable fluctuation of domains, mainly the C-terminal domain relative to the / V-terminal domain, which causes a slight opening / closing of the protein. The results showed that these fluctuations lead to a weakening of the bound ligand interactions. The dynamics of the inhibitors themselves in the binding site were also investigated. The results of experiments on the transmitted NOE effect and simulations of the molecular dynamics of MurD ligase in complex with inhibitors have shown that individual parts of those inhibitors that interact with all three domains are moving in the binding site. This motility is also related to protein flexibility.

Based on our results and an overview of the known crystal structures of Mur protein complexes with natural substrates, we selected the binding site of the UDP of a portion of the substrate. Choosing this binding site has the following advantages:

- The ligand is primarily in contact with the // - terminal domain but has no contact with the Cterminal domain. Therefore, the N- and C-terminal fluctuations are not affected by the binding.

- The UDP portion of the substrate is structurally identical to the MurA to MurG enzymes, meaning that the inhibitors thus designed could potentially inhibit a very wide range of Mur enzymes. The bonded conformation and interactions of the UDP of the substrate are also very similar.

- The selected binding site is slightly smaller and is suitable for anchoring molecules similar to hydrogen compounds. This means that there is more chance of finding a well-fitting compound with a virtual grid. The entire binding site of the MurD protein substrate is large and quite flexible, which would complicate the virtual lattice process.

b) The course of virtual gratings

Virtual caching was performed using Glide version 5.7 (Glide, version 5.7, Schrodinger, LCC, New York, NY, 2011), which is part of the Schrodinger suite 2011. Virtual caching was performed on an HP Elite 7100 workstation with an operating system Ubuntu 10.04 and an Intel Core i7 860 processor with eight cores at 2.80 GHz. In the first phase, we anchored a series of Clean lead like subset compounds, obtained from the freely accessible ZINC database (Version 11) (available at http://zinc.docking.org/). The series contained 1590768 molecules. We chose the MurA protein for the first target because we were looking for compounds that would potentially work on a wide range of Mur proteins. It is desirable that the inhibitors work as much as possible at the start of the chain leading to bacterial peptidoglycan biosynthesis. This stage served primarily to secrete poorly binding molecules. Due to the very large number of molecules in Glide, we have chosen the option of high throughput Virtual screening in Glide (HTVS), which enables the virtual grid to be executed in an acceptable time. The 10,000 molecules with the best binding to MurA (we used the Gscore evaluation function in the Glide program as a criterion) were then anchored with standard precision (the "SP - standard precision" option in the Glide program) to MurA proteins (PDB code 1UAE), MurB (PDB code 2MBR), MurC (PDB code 1P3D), MurD (PDB code 2UAG), MurF (PDB code 2AM1), and MurG (PDB code 1NLM). We selected the best 100 compounds for each of these proteins according to Gscore's evaluation function. Thus, we obtained 600 molecules, which were then anchored with the highest precision (the "XP - extra precision" option in the Glide program) to MurA proteins (PDB code 1UAE) and MurD (PDB code 2UAG). For each of these proteins, we selected the top 20 compounds according to Gscore's evaluation function. Thus, we obtained 40 compounds that were most anchored to the proteins MurB (PDB code 2MBR), MurC (PDB code 1P3D, MurF (PDB code 2AM1) and MurG (PDB code 1NLM). further bioassays The test compounds were purchased from various manufacturers as indicated in Table 1.

Biological tests

1. Principle

The MurD ligase catalyzes the reaction where a UDP-MurNAc-L-Ala-D-Glu (UMAG) product is formed from the UDP-MurNAc-L-Ala (UMA) substrate. ATP is involved in the reaction, which decomposes into ADP and inorganic phosphate. The formation of inorganic phosphate is determined by following the formation of a green colored complex with malachite green dye (Biomol Green reagent). The activity of the enzyme is proportional to the amount of phosphate produced. This should be within the linearity of the phosphate calibration curve. For compounds with inhibitory activity, the amount of phosphate produced is reduced compared to the negative control, where we do not have inhibitory activity. The inhibitory activity is expressed by residual activity (RA) and constant IC50.

2. Reagents

MurD enzyme: isolated and completely purified from E. coli strain BL21 (DE3) pLysS, following the procedure described in the literature (Simcic, M .; Hodosček, M .; Humljan, J .; Kristan, K .; Urleb, U. ; Kocjan, D.; Golič Grdadolnik, SJ Med. Chem. 2009, 52, 2899-2908). The enzymes are stored at -24 ° C. It is diluted with enzyme dilution buffer before use.

Enzyme Dilution Buffer:

mM Hepes, pH 8 (adjusted by the addition of HC1 solution) mM dithiothreitol (DTT)

The reaction mixture:

- 50 mM Hepes, pH 8

- Bi-distilled water (milliQ)

- 0.01% Triton-X114

- 10mMNH ₄ SO ₄

- 5 mM MgCl ₂ 100pMD-Glu

- 80 μΜ UMA

- 0.4 mM ATP

Inhibitoi:

Prepare 10 mM stock solution of inhibitors in DMSO. In case of poor solubility of the compounds in DMSO, 5 mM and 2 mM concentrations are used, respectively. The concentration of DMSO in the test solution relies on 5% (v / v).

3. The procedure or. Workflow

The tests are performed on microtiter plates. Pipette 2.5 pL of appropriate inhibitor dilution in DMSO (2.5 μ (DMSO in case of control), 32.5 pL of reaction mixture and 15 pL of MurD enzyme solution (15pL of enzyme dilution buffer in background control) into each well. The microtiter plates were incubated at 37 ° C with the reaction mixture and the reaction was stopped after 15 min by the addition of 100 µL of Biomol Green reagent. After 5 min, measure the absorbance at 650 nm on a PowerWave XS MQX200R2 microtiter reader (BioTek Instruments, Inc.)

Determination of residual activity (RA) of the MurD enzyme:

Residual enzyme activity is defined as the ratio of the catalytic activity of an enzyme to the presence of an inhibitor and no inhibitor. We calculate it according to the following equation:

RA (%) - (Al - A _s and _ep al) / (Ak ^_ Asiepa) ^x 100

Ai - absorbance of the reaction mixture with the enzyme and inhibitor at 650 nm

Asiepai - Absorbance of an enzyme-free reaction mixture with an inhibitor at 650 nm (background absorbance)

Ak - absorbance of the reaction mixture with enzyme and no inhibition at 650 nm

Asiepa - absorbance of the reaction mixture without enzyme and without inhibition at 650 nm

Determination of constant IC50

IC50 constant means the inhibitor concentration that causes the rate of MurD catalyzed reaction to be reduced by half. It is determined at 80 μΜ UMA concentration and 100 μΜ d-G1u concentration. The procedure determines the RA of the MurD enzyme at 7 different inhibitory concentrations. For each inhibitor concentration, expressed in μΜ, we calculate its decimal logarithm. Draw a graph where, on the x axis, the decimal logarithm of the inhibitor concentration on the y axis is the residual activity of the enzyme. The IC50 constant is determined by fitting the curve with nonlinear regression. The equation of the RA (%) curve, depending on the decimal logarithm of concentration, is described by the equation:

RA (%) = RA (%) _DN0 + (RA (%) top - RA (%) _DNO ) / (l + 10 ^(logIC5 ° - ^logc) )

RA (%) bottom - Residual activity at the bottom of the curve

RA (%) top - Residual activity at the top of the curve

Loge - the decimal logarithm of the logICso inhibitor concentration - the decimal logarithm of the IC50 constant

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Tabelal: Results of bioassays to determine the efficacy of MurD ligase inhibitors.

The results are given as RA (%) at a given inhibitory concentration indicated in parentheses (the maximum concentration at which the inhibitor is still soluble in the reaction mixture), or as a constant IC50. The results show that compounds I and II are potent inhibitors of MurD ligase and are therefore suitable for the development of agents for the treatment of bacterial infections.

ZINC code Manufacturer, manufacturer's code Structure RA (%) 06493851 Asinex BAS03848012 (TV Y ° H OO 1-1--7 / \\ ^H \ = / RA 12% (100 μΜ) IC ₅ o = 17 μΜ 17992791 Vitas-M STL150455 fi T iT ^h 0 0 RA 10% (25 μΜ) IC ₅₀ = 5 μΜ 09421208 Vitas-M STK651077 OMe ^ ⁷ ϊ I 1 _N ^h OON ^H \ = / RA 100% (5 μΜ) 8604403 ChemDiv 720-0594 c, O Hhl RA 100% (50 μΜ)

12533064 Enamine T0507-3131 CO ...... t o RA 100% (10 μΜ) 2922855 Asinex BAS00340037 nh ₉ 5% Me ⁰ RA 100% (500 μΜ) 13154275 Asinex BAS00489498 ° T RA 100% (500 μΜ) 22966418 Enamine T6657437 oh U N RA 100% (500 μΜ) 47501494 Enamine T6865610 Me ^ N ^ N RA 100% (500 μΜ) 4247364 Enamine T5832969 OMe hn- ^ v ⁿ x / A J 1 / ^- s 0:? 'V' Me RA 100% (500 μΜ)

20718371 IBScreen STOCK6S-67067 Me F ^N \ ° t> ° ^H RA 100% (500 μΜ) 67721 IBScreen STOCK2S-20923 OH Λ ^0H ην ^χΧ > τ ^ν \ H oV-h-Mjl Me RA 100% (500 μΜ) 32221280 IBScreen STOCK6S-93462 0 o n '^ i Me RA 100% (500 μΜ) 22049850 Toronto Research Chemicals B208090 H / = \ / ° V ^ ^0H »- = ⁷ 0 0 HO RA 100% (500 μΜ) 67725 Vitas-M STK559539 - ~ -OH 0 r— <. JI OH HN '^ Xr ^N \ H oV-UmO Me RA 100% (500 μΜ)

13320180 Vitas-M STK478736 0 Ci HN ' Χ-Χ- Ό -N RA 100% (500 μΜ) XX .OH Me. 8991067 Vitas-M STK617380 0. ..0 0 RA 100% (500 μΜ) Me. .N. / NH X v Me H -N θΧ N- ~ G 44973917 Enamine T6564728 .N ° 0 RA 100% (500 μΜ) U H- o- • Et 13691487 IBScreen STOCK6S-47703 ζ ^Ν νΛ Λθ RA 100% (500 μΜ) < ^with X> = 0 H

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Patent claims

Claims (6)

Compounds I and II and their pharmaceutically acceptable salts II for inhibition of MurD ligase. Compounds I and II according to claim 1, for use in inhibiting bacterial growth. Compounds I and II according to any one of claims 1 or 2 for use in the treatment of bacterial infections, preferably by inhibition of MurD ligase. 4. Pharmaceutical forms containing compound I and / or compound II and / or their pharmaceutically acceptable salts according to any one of claims 1 to 3, for inhibiting MurD ligase. 5. Pharmaceutical forms according to claim 4 for use in inhibiting bacterial growth. Pharmaceutical forms according to any one of claims 4 or 5 for use in inhibiting bacterial growth, preferably by inhibition of MurD ligase.