Derivatives of benzimidazole sulfonamide compounds and use thereof

Abstract

The present invention relates to benzimidazole sulfonamide compounds. The proposed compounds can be used as inhibitors of the CCR7 receptor and, in particular, of the CCL21 ligands for the chemokine receptor.

Description

[0001] Benzimidazole sulfonamide derivatives and their applications

[0002] Application area

[0003] The present invention relates to benzimidazole-sulfonamide compounds. The proposed compounds can be used as inhibitors of the CCR7 receptor, in particular, ligands for the chemokine receptor CCL21.

[0004] Prior art

[0005] Currently, oncological diseases are one of the main causes of death. Among oncological diseases, lung cancer is one of the most common types, along with breast cancer and colon and rectal cancer.

[0006] In the structure of this disease, up to 90% belong to non-small cell lung cancer (NSCLC), which is characterized by the worst prognosis.

[0007] Neoplasms in this case arise from the growth of tumor stroma by vessels and cell infiltration. These neoplasms are often associated with the formation of chemokines. Chemokines are a family of soluble proteins that control the migration of leukocytes under physiological conditions and during inflammation. Chemokines are defined according to a systematic nomenclature based on the position of the amino acid residues cysteine ​​near the N-terminus of the polypeptide chain of the protein. Described

[0008] 4 types of such arrangement. There are 4 subfamilies of chemokines: CC, CXC, CX3C and CS. The CXC subfamily, in turn, is divided into. CXC- ELR+ and CXC-ELR- depending on the presence or absence of a section of the amino acid sequence consisting of glutamic acid. leucine and arginine (ELR motif). Both chemokine receptors and their ligands are synthesized in endothelial, epithelial and immune system cells, fibroblasts and keratinocytes. For some of them, this is a constant process, while others are formed as a result of changes in the cell microenvironment and are therefore inducible.

[0009] For example, tumor necrosis factor-α (TNF-α) increases the production of the chemokine CCL5 and its receptor CCR5 in cells of the central nervous system, while interferon-gamma (INF-γ) is a potent inducer of CXC-ELR chemokine expression. Among others, chemokines of the CCL21 type regulate lymphocyte migration and bind to the CCR7 receptor, which is expressed by naïve T cells and dendritic cells. These chemokines are important for activating the local antitumor immune response, including the activation of dendritic cells, the mobilization and activation of naïve T cells, and the formation of lymphoid structures. Similar to other neoplasms, in NSCLC, CCL21 expression is important for the formation of lymph node-like structures associated with tumor tissue. Such structures may be responsible for patient survival. and in experimental lung cancer - tumor reduction.Ex vivo studies conducted with cancer cells have shown that the antitumor effect of high CCL21 expression is mediated by the activation of dendritic cells and the release of CXCL9 and CXCL10, which inhibit angiogenesis. Thus, modulating CCL21 expression may be a possible therapy for lung cancer, in particular non-small cell lung cancer.

[0010] In general, CCR7 and CCL21 receptor are associated with breast cancer, head and neck cell carcinoma, esophageal cancer, chronic lymphocytic leukemia, mantle cell lymphoma, fibrotic disorders. In all these cases, metastasis formation, actin polymerization, angiogenesis and, as a result, poor prognosis for survival are noted.

[0011] Currently, antibodies, such as those described in patent WO 2007 / 003426, or antibody conjugates, such as those disclosed in WO 2018 / 142322, are used as inhibitors against CCR7. However, antibodies and their conjugates can be difficult to obtain or have a low yield, are unstable in storage, and their use is complicated by allergic and other side effects.

[0012] Thus, it is of interest to search for alternative drugs and compounds that could potentially be used as inhibitors of the chemokine receptors CCR7 and CCL21.

[0013] Brief description of the invention

[0014] The present invention provides compounds of formula 1

[0015] R1-R7 may each and independently represent radicals selected from hydrogen, C1-C6 alkyl, C1-C6 alkyloxy, C1-C6 alkyloxyhydroxyl, C1-C6 alkylamide, C1-C6 alkyl(C3-C6 cycloalkyl) hydroxyl, aminocarboxyl, C1-C3 alkylaminosulfonic acid, C1-C3 alkylsulfonamide. Preferably, the compound is selected from the following:

[0016] 

[0017]

[0018] The compounds of formula 1 can be used as an inhibitor of the chemokine receptor CCR7. In particular, these compounds can be used as inhibitors of the receptor CCL21.

[0019] In addition, a pharmaceutical composition is proposed containing a compound of formula 1 in a therapeutically effective amount. This composition may also include pharmaceutically acceptable auxiliary substances. The dosage of the compound of formula 1 may vary from 0.1 nM to 10 mg.

[0020] It is also proposed to use the compound of formula 1 in a therapeutically acceptable amount for the treatment of breast cancer, esophagus, lung cancer, and fibrotic disorders.

[0021] Description of figures

[0022] Fig. 1 includes an IR spectrum of a compound according to the invention.

[0023] Fig. 2 includes the NMR spectrum of the compound according to the invention.

[0024] Fig. 3. Migration assessment. 1 control, against FBS10%, 2- CC 21 against compound 1a. Detailed description and inventions

[0025] The present invention provides compounds of formula 1

[0026] R1-R7 can each and independently represent radicals selected from hydrogen, C1-C6 alkyl, C1-C6 alkyloxy, C1-C6 alkyloxynhydroxyl, C1-C6 alkylamide, C1-C6 alkyl(C3-C6-nicloalkyl)hydroxyl, aminocarboxyl, C1-C3 alkylaminosulfonic acid, C1-C3 alkylsulfonamide.

[0027] Preferably, the compound is selected from the following:

[0028] 

[0029]

[0030] Compounds of formula 1 can be used as an inhibitor of the chemokine receptor CCR7. In particular, these compounds can be used as inhibitors of the receptor CCL2L

[0031] The proposed compounds can be in the form of a pharmaceutical composition. Such a composition: contains a compound of formula 1 or any of the proposed compounds, alone or in combination, in a therapeutically effective amount. The term "therapeutically effective amount" is an amount that alleviates one or more symptoms of cancer, or cures it. The term "cancer", "cancer disease" or "oncological disease" are used interchangeably and mean predominantly breast cancer, esophageal cancer, lung cancer, fibrotic disorders. Fibrotic disorders can be a disorder selected from the group consisting of pulmonary fibrosis, chronic obstructive pulmonary disease, liver fibrosis, rheumatoid arthritis, congestive heart failure, chronic kidney disease. hypersensitivity pneumonitis.respiratory bronchiolitis / interstitial lung disease, Schistosoma Munson infection, primary pulmonary hypertension due to plexiform lesions, pulmonary manifestations of herpes virus-related diseases, dermatologic manifestations of herpes virus-related diseases, keloid scars, lupus, nephrogenic fibrosing dermopathy, fibrotic lesions associated with Schistosoma japonicum infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal remodeling in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis, congestive heart failure and other postischemic conditions, scarring after abdominal surgery, scarring after trabeculotomy in open-angle glaucoma, and any combination of these diseases. The pharmaceutical composition may also include pharmaceutically acceptable excipients.The types of such substances are described in the guidelines on drug technology.

[0032] (for example, "Technology of manufacturing medicines"

[0033] V. A. Grossman, Geotar-Media, 2018). The content of the compound of formula 1 is determined by the therapeutically effective amount: and in general can vary from 0.1 nM to 10 mg. in each individual dosage form.

[0034] The possible content and the prescribed dose for the patient is determined by the clinician and depends on many factors, such as the severity of the disease, the duration of the course, polypharmacy, the patient's condition and the presence of concomitant diseases. The duration of admission can also be determined by the doctor, including from the risk-benefit ratio and therapeutic response.

[0035] The present invention will now be illustrated by examples, which are not intended to define the scope of the invention.

[0036] Example 1

[0037] Method for obtaining compounds of formula 1

[0038] Compounds of formula 1 were obtained according to the scheme below.

[0039] In general, the preparation of sulfonamide by reaction of sulfonyl chloride with an amine and subsequently the reaction of hydroxybenzimidazole with chlorosulfonic acid was used.

[0040] The general scheme is presented below.

[0041] The following starting materials were used for the preparation: the corresponding derivative of 2-naphthylamine and the derivative of 2-oxo-benzimidazole-5-chlorosulfonic acid.

[0042] Compound 1a of the following structure was used as a representative one: 1-naphthylamino-2-oxobenzimidazole-5-(1-naphthyl)sulfonamide; (N-(2-naphthyl)-2-oxo-2 ; 3-dihydro-1H-benzimidazole-5-sulfonamide' N-naphthalen-2-yl-2-oxo-1 ; 3-dihydrobenzimidazole-5-sulfonamide)

[0043] The starting materials were loaded into a 500 ml flask: 3-naphthylamine and sulfochloride in a ratio of 2-naphthylamine in excess of 1:1.2 with respect to chlorosulfonic acid, water was used as a solvent; The synthesis was carried out at an elevated temperature of up to 60 ° C, for 3 and. Then, the resulting mixture was purified by crystallization. At the last stage, a derivative of sulfanilamide was obtained using derivatives of chlorosulfonic acid in a flask, with rapid heating to 130-145 º C, stirring for 3 hours. After cooling the mixture, quickly drop it into 1 kg of ice. After the ice has melted, filter the resulting compound, which has precipitated in the form of crystals, wash with ice water until the washings are neutral, and then add 400 ml of cooled 1-naphthylamine or its derivative, and stir for 30 minutes.

[0044] Next, after this, the mixture is cooled to 0°C, mixed with ice, and while stirring, acidified with hydrochloric acid to pH 2. The mixture is kept in the refrigerator, the precipitated crystals are filtered and recrystallized from water using activated carbon. The target product is obtained in the form of plates or crystals.

[0045] The final product was the target compound, the yield was on average 65-70%.

[0046] The obtained compounds were analyzed by IR and NMR spectroscopy to confirm the structure; the data are presented in the Figures.

[0047] NMR spectra were obtained on a Broker Avance II 300 spectrometer at 300 MHz (1H) and 75 MHz (13C) in D2O in pulsed mode followed by Fourier transformation using Me4Si as an internal standard. Spin multiplets are designated as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet).

[0048] IR spectra in the solid phase were recorded on a Broker Tensor-27 instrument with a low total internal reflection module.

[0049] The IR spectra of the obtained compound 1a are shown in Fig. 1.

[0050] The NMR spectrum of the obtained compound 1a is shown as a representative one in Fig. 2.

[0051] Example 2

[0052] Analysis of biological activity

[0053] Compounds 1a were used to analyze biological activity. The analysis was performed on melanoma cells isolated from surgical material of patients admitted for treatment to the Pirogov Russian National Research Medical University of the Ministry of Health of the Russian Federation. Cell cultivation was carried out using the RIFresbney method. The commercially available drug Human FibrOut 9 was used as a selective agent that prevents fibroblast growth in primary culture. The cells were cultured continuously for at least 9 passages, the last passage was used for the study. The cells were planted in special inserts with a pore diameter of 8 μm in Matrigel. Tumor cell migration was analyzed using the method described in the guidelines of A. Yu. Aleksandrova (2008). To assess migration processes, an automatic system for monitoring living cells Cell-IQ was used. DMEM nutrient medium was added to each well, Matrigel was added to cover the bottom.The plates were left in an incubator for 1 hour in a carbon dioxide environment at a temperature of 37. º C. Next, background measurements were performed. After that, compounds were added in saline solution in various concentrations. The resulting migration was measured visually, by counting cells in the field of view. The CCL21 receptor was used as a control. Table 1

[0054] The following data were obtained:

[0055] Migration Control towards FBS10%

[0056] 218 cells

[0057] CCL21 vs. compound 1a 87 cells

[0058] The data on migration control are presented in Fig. 3.

[0059] Next, to assess the activity, migration was assessed at different concentrations Table 2

[0060] As can be seen, with an increase in concentration, the efficiency of inhibition of cell migration increases, which indicates an increase in effectiveness.

[0061] Example 3

[0062] Analysis of inhibitory activity against the chemokine receptor CCR7

[0063] To predict possible pharmacotherapeutic effects and mechanisms of action of the compounds according to the invention, the PASS Online web service was used. The compounds selected with the given activity are presented in Table 3.

[0064] Table 3. Compound 1a with toxic dose and OECD classification of chemicals for acute toxicity to rodents

[0065] IP - intraperitoneal route of administration, IV - intravenous route of administration. Oral - oral route of administration, SC - subcutaneous route of administration.

[0066] In addition to therapeutic efficacy, an important parameter is the safety of the drug. In order to determine the latter, a computational assessment of the acute toxicity of the selected compounds was carried out using the GUSAR Online Acute Toxicity web service. This service predicted the LD values 50in rats with different routes of administration (intraperitoneal, intravenous, oral and subcutaneous routes of administration). Table 3 also presents the results of the prediction of LD values 50 for compounds with predicted chemokine receptor inhibitory activity.

[0067] From the data presented in Table 3 it is evident that the studied compounds are: non-toxic or: low-toxic compounds.

[0068] The binding of compounds to biological targets was also assessed using molecular docking,

[0069] Molecular docking is based on the assessment of ligand binding to the receptor region and allows to obtain information about its structural features, including conformations, orientation, and organization in the receptor site [L. Ferreira, R. dos Santos, G. Oliva, and A. Andricopulo, "Molecular docking and structure-based drag design strategies," Molecules, vol 20, no. 7, pp. 13384–13421 , 2015]. Chemokine receptors belong to the GPCR group of receptors. Receptors of this group activate the cellular response through interaction with cognate G proteins. It was found that similar interactions are observed for the Vercimon ligand in the chemokine receptor CCR9 [5]. The key fragment in the case of Vercimon is the sulfone group, which forms hydrogen bonds with the amino groups of the main chain Glu322, Arg323 and Phe324, as well as with the side chain, in particular with Tyr317. These amino acids play a key role in the formation of the allosteric binding site of CCR9.

[0070] Compound 1a was considered as an antagonist for the orthosteric binding site in the chemokine receptors CC R.7 and CCR9, since it contains pharmacophore fragments similar to those of the antagonists Cmp2105 and Vercimon, namely: a five-membered ring with nitrogen atoms and a sulfone or sulfonamide group. In addition, the presence of several phenyl rings in the structure additionally promotes the formation of -π-π- and or •π- catone stacking interactions similar to those of the reference ligands. All the studied compounds were subjected to the molecular docking procedure in the binding sites of CCR7 and CCR9.

[0071] Preparation of ligands and receptor

[0072] Crystallographic structures of the CCR7 receptor in complex with the antagonists Cmp2105 (PDB code 6QZH [9]) and the CCR9 receptor in complex with

[0073] Vercimon. (PDB code 5LWE

[0010] ), for modeling the binding process. were downloaded from the non-commercial: Protein Data: Bank database

[0011] .

[0074] The geometric parameters of each receptor were prepared for calculations as follows: hydrogen atoms were added and minimized, water molecules and other low-molecular structures were removed, except for reference compounds, the entire structure was limitedly optimized: using the OPLS3e

[0012] force field method at physiological pH values ​​of the environment.

[0075] The geometric parameters of potential ligands were collected into a single database and optimized taking into account all acceptable conformations.

[0076] Binding site analysis

[0077] In both chemoreceptors, intracellular orthosteric binding sites of antagonists were examined. Compound 1a was subjected to the molecular redocking procedure in order to evaluate the reproduction of the geometric parameters of the ligands in their binding sites. In both cases, the RMSD (root mean square deviation) values ​​do not exceed 1 A 2, which indicates the correctness of the selected docking protocol. As functional amino acids for binding antagonists, Thr93, Arg94, Val331 Lys332 and Phe333 can be considered - with which the studied antagonist Str2105 forms hydrogen bridges for CCR7.

[0078] For CCR9, Arg323, Phe324 - hydrogen bonds can be considered,

[0079] Arg 144 and Arg323 are salt bridges, Tyr3.1.7 are π-π-stacking interactions between the aromatic RING of the amino acid and the chlorophenyl and phenyl rings of the ligand. The amino acid numbering corresponds to PDB codes 6QZH and 5LWE.

[0080] Molecular docking

[0081] Ligand–receptor complexes were subjected to a procedure of constrained minimization of ligand-induced changes within the binding site taking into account the charge characteristics of the surrounding amino acids using quantum chemical calculations. The compounds were docked using the ligand forced positioning (1FD) protocol with the following conditions: flexible protein and ligand, grid matrix size is 15 Å, amino acids within 5 Å of the ligand are constrainedly optimized taking into account the ligand effect. The ranking of the docking solutions was performed by evaluating the following computational parameters: docking score (based on the GlideScore minus penalties), ligand efficiency (LE, which takes into account the per-atom distribution of the scoring function), and the model energy value parameter (Emodell), which includes the GlideScore value, the energy of unbonded interactions, and the parameters of the energy spent on folding the compound into the binding site.

[0082] Results of the assessment of binding of compounds to biological targets.

[0083] The results of molecular docking at the above-mentioned binding sites of the antagonist and CCR-7 receptor for the lead compounds are presented in Table 4.

[0084] Table 4. Molecular docking results of CCR7 receptor binding Compound 1a showed high binding in the CCR7 receptor and in the CC.R9 receptor. Remarkably, compound 1a binds in the site of both receptors. Compound 1a in both sites forms similar interactions: hydrogen bridges and - π- πc-stacking interaction with the aromatic ring of tyrosine. In the CCR7 receptor, compound 1a forms a hydrogen bridge between the oxygen atom and the amino group of arginine, and in the CCR9 receptor, the oxygen of the sulfonamide group participates in the formation of a similar bond. In addition, additional bridges are formed between the oxygen of the sulfonamide bond and the amino group of phenylalanine and the cyclic nitrogen atom with the hydroxyl group of asparagine.

[0085] The interaction with Arg154 and Tyr326, located in the cytoplasmic cleft part (opened upon activation of GPCR) in the CCR7 receptor, as well as with the main chain amino acids (Arg323, Phe324, Asp327) in the CCR9 receptor indicates that compound 1a can effectively bind to the antagonist site similar to the ligands: Str2105 and Vercimon, Example 3

[0086] Assessment of cell migration.

[0087] All compounds were tested for efficacy according to the experiment described in Example 2.

[0088] Data on the compounds according to the invention are presented in the table below. 5; .

[0089] |

[0090] |

[0091] |

[0092] |

[0093] As follows from the experimental results, all compounds have similar inhibitory activity.

Claims

CLAUSE OF INVENTION 1. Compound of formula 1 R5 selected from the following:  2. The compound according to claim 1 for use as an inhibitor of the chemokine receptor CCR7.

3. The compound according to I. 1, characterized in that it is a compound 1a 4. A pharmaceutical composition containing the compound according to claim 1 in a therapeutically effective amount.

5. A pharmaceutical composition according to item 4, additionally including pharmaceutically acceptable excipients.

6. A pharmaceutical composition according to claim 4, containing from 0.1 nM to 10 mg of the compound according to claim 1.

7. Use of a compound according to claim 1 in a therapeutically acceptable amount for the treatment of breast, esophageal, and lung cancer.

8. Use of a compound according to claim 1 as an inhibitor of the chemokine receptor CCR7.

9. The use according to claim 8, characterized in that the chemokine receptor is a CCL21 receptor.

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