Indolenaphthalene acetamide derivatives and use thereof

Abstract

The present invention relates to indolenaphthalene acetamide derivatives and the use thereof. The proposed compounds can be used as inhibitors of the CCR7 receptor and, in particular, of the CCL21 ligands for the chemokine receptor and, therefore, as a therapeutic agent in the case of cancerous diseases.

Description

[0001] Indolenaphthaleneacetamide derivatives and their applications

[0002] Scope of application

[0003] The present invention relates to indolenaphthaleneacetamide derivatives and their use. The proposed compounds can be used as CCR7 receptor inhibitors, in particular, ligands for the chemokine receptor CCL21.

[0004] Prior art

[0005] Today, oncological diseases make a large contribution to the causes of human mortality, including young and middle-aged people. 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 lung cancer, up to 90% belong to non-small cell lung cancer (NSCLC), which is characterized by the worst prognosis for survival. In this case, neoplasms originate 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 protein polypeptide chain. Four types of such arrangement have been described. There are four subfamilies of chemokines: CC, CXC, CX3C and XC. The CXC subfamily, in turn, is subdivided into CXC- ELR+ and CXC-ELR- depending on the presence or absence of a region 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 cells, fibroblasts, and keratinocytes. For some, this is a continuous process, while others are formed as a result of changes in the cell microenvironment and are therefore inducible.

[0007] For example, tumor necrosis factor (TNF-a) increases the formation of the chemokine CCL5 and its receptor CCR5 in cells of the central nervous system, while interferon-gamma (INF-y) 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 naive T lymphocytes and dendritic cells. These chemokines are important for the activation of the local antitumor immune response, including the activation of dendritic cells, the mobilization and activation of naive T lymphocytes, 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, for tumor shrinkage.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, particularly non-small cell lung cancer.

[0008] 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, actin polymerization, angiogenesis and, as a result, poor prognosis for survival are noted.

[0009] 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 adverse reactions.

[0010] 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.

[0011] Brief description of the invention

[0012] The present invention provides the following compounds:

[0013]

[0014] In a preferred embodiment, a connection is proposed

[0015] The compounds listed above can be used as an inhibitor of the chemokine receptor CCR7. In particular, these compounds can be used as inhibitors of the receptor CCL21.

[0016] In addition, a pharmaceutical composition is proposed, containing any compound given above, or a combination thereof, in a therapeutically effective amount. This composition may also include pharmaceutically acceptable excipients. The dosage of the compound of formula 1 may vary from 0.1 nM to 10 mg.

[0017] It is also proposed to use the compound of formula 1 in a therapeutically acceptable amount for the treatment of breast cancer, esophagus cancer, lung cancer, and fibrotic disorders.

[0018] Description of figures

[0019] Fig. 1 includes an IR spectrum of a compound according to the invention.

[0020] Fig. 2 includes the NMR spectrum of the compound according to the invention.

[0021] Fig. 3. Migration assessment.

[0022] 1 - control, against FBS10%,

[0023] 2- CCL21 vs. compound 1a.

[0024] Detailed description of the invention

[0025] Preferably, the compound is selected from the following:

[0026] A composition containing the compounds listed above in a therapeutically effective amount is also provided.

[0027] The term "therapeutically effective amount" is an amount that results in relief of one or more symptoms of, or cure of, cancer. The term "cancer," "cancer disease," or "oncological disease" are used interchangeably and include, but are not limited to, breast cancer, esophageal cancer, lung cancer, fibrotic disorders. The fibrotic disorder may 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 restructuring in pancreatitis and stromal fibrosis, uterine fibroids, ovarian fibrosis, corneal fibrosis, congestive heart failure and other postischemic conditions, scarring after abdominal surgery, scarring after trabeculae otomena in open-angle glaucoma, and any combination of these diseases.

[0028] This pharmaceutical composition may also include pharmaceutically acceptable excipients. The types of such substances are described in the guidelines on drug technology (e.g., "Technology of Manufacturing Drugs", V.A. Grossman, Geotar-Media, 2018). The content of the compound of formula 1 is determined by the therapeutically effective amount and can generally vary from 0.1 nM to 10 mg in each individual dosage form.

[0029] The possible content and the prescribed dose for the patient are determined by the clinical physician and depend 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 administration can also be determined by the physician, including from the risk-benefit ratio and therapeutic response.

[0030] The compounds listed above can be used as inhibitors of the chemokine receptor CCR7. In particular, these compounds can be used as inhibitors of the receptor CCL21, since CCR7 is a receptor for two ligands: the chemokines CCL19 and CCL21.

[0031] In addition, a pharmaceutical composition is proposed, containing any compound given above, or a combination thereof, in a therapeutically effective amount. This composition may also include pharmaceutically acceptable excipients. The dosage of the compound of formula 1 may vary from 0.1 nM to 10 mg.

[0032] It is also proposed to use a compound of formula 1 in a therapeutically acceptable amount for treating breast cancer, esophagus, lung cancer, and fibrous disorders. The CCR7 receptor is found on various cancer cells, and the expression of these receptors on cancer cells is associated with their metastasis to lymph nodes. Thus, the compounds according to the present invention are inhibitors of the CCR7 receptor and can be used for the therapy of various types of cancer, including for the treatment of breast cancer, esophagus, lung cancer, and fibrous disorders.

[0033] The present invention will now be illustrated by examples, which are not intended to define the scope of the invention.

[0034] Examples

[0035] Example 1

[0036] Method for obtaining compounds of formula 1

[0037] Compounds of formula 1 were prepared according to the general scheme below. The method was modified depending on the derivatives obtained.

[0038] Introduction of protective groups

[0039] Getting the target connection

[0040] In the general case, m-chloronaphthalene acetyl chloride was used, the reaction yielded m-chloronaphthalene acetamide chloride, then the resulting acetamide chloride was reacted with 4-aminophthalimide (1,3-dioxoisoindol-5-yl-2-naphthylamine). The target compound was obtained by removing the protection in the form of a chloro group.

[0041] To obtain 4-aminophthalimide, 4-nitrophthalimide was obtained from 1,3-bioxoisoindole-2-naphthylamine. 45 g of nitric acid were added to 324 g of sulfuric acid with stirring at a temperature of 0-2 °C. Then, 30 g of phthalimide were added to this mixture at a temperature of 2-5 °C, stirring carefully. Then, the reaction mass was left for 5 hours, monitoring the completion of the reaction by chromatography. The temperature of the mixture slowly rose from 5 °C to 25 °C while maintaining. The temperature was reduced by adding water and ice with stirring. The resulting suspension was filtered and washed with ice water to a pH of 4. The isolated pasty substance was dried at 40-50 °C, obtaining 31 g of 4-nitrophthalimide.

[0042] Next, a suspension of 5.8 g of 4-nitrophthalimide and 30 ml of 25% ammonia solution was added with stirring to a solution obtained from 150 ml of water and 25 g of sodium dithionite. The reaction mixture was stirred for 4 hours at normal temperature to complete the reduction reaction; monitoring was performed using chromatography. A slightly alkaline pH was maintained. The amine suspension was filtered and the isolated product was dried at 40-50°C. About 3 g of 4-aminophthalimide were obtained.

[0043] Next, m-chloronaphthaleneacetyl chloride was obtained by chlorinating naphthalene with chlorosulfonic acid.

[0044] The starting materials were loaded into a 500 ml flask: m-chloronaphthaleneacetchloride 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 hours. Then, the resulting mixture was purified by crystallization. At the last stage, a sulfanilamide derivative was obtained using chlorosulfonic acid derivatives in a flask, with rapid heating to 130-145 ° C, stirring for 3 hours. After cooling the mixture, it was quickly dropped into 1 kg of ice. After the ice melts, the resulting compound m-chloronaphthaleneacetchloride, which precipitated in the form of crystals, is filtered off, washed with ice water until the washings are neutral.

[0045] To obtain the target compound, 4 g of m-chloronaphthaleneacetyl chloride and 3 g of 4-aminophthalimide were mixed in a flask containing a solvent of tetrahydrofuran and triethylamine. The resulting mixture was stirred for 20 min. The completion of the reaction was monitored by chromatography. Thus, about 3 g of N-(1,3-dioxoisoindol- yl)-2-naphthalen-1-ylacetamide were obtained.

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

[0047] NMR spectra were obtained on a Bruker 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 multiplicities are denoted as s (singlet), d (doublet), t (triplet), q (quartet), or w (multiplet).

[0048] Solid-phase PC spectra were recorded on a Bruker Tensor-27 instrument with a low total internal reflection module.

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

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

[0051] Example 2

[0052] Analysis of biological activity

[0053] Compounds 1a were used for the analysis of biological activity.

[0054] The analysis was performed on skin 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 performed using the RIFreshney method. The commercially available Human FibrOut 9 drug 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). The migration processes were assessed using the Cell-IQ automated system for monitoring living cells. DMEM nutrient medium was added to each well, and Matrigel was added to cover the bottom. The plates were left in an incubator in a carbon dioxide environment at a temperature of 37 °C for 1 hour.Next, background measurements were performed. After that, compounds were added in saline 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.

[0055] Table 1

[0056] The following data were obtained:

[0057] IC 50 Raw264.7 3.8x10' 5 M 1C 50 A549 4.1 X10' 5 M

[0058] Migration Control towards FBS10%

[0059] 256 CCL21 cells vs. compound 1a 85 cells

[0060] The data on migration control are presented in Fig. 3.

[0061] Next, to assess the activity, migration was assessed at different concentrations.

[0062] Table 2

[0063] As can be seen, with increasing concentration, the efficiency of inhibition of cell migration increases, which indicates an increase in efficiency.

[0064] Example 3

[0065] Analysis of inhibitory activity against the chemokine receptor CCR7

[0066] 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.

[0067] Table 3. Compound 1a with toxic dose and OECD classification of chemicals for acute toxicity to rodents. IP - intraperitoneal route of administration, IV - intravenous route of administration, Oral - oral route of administration, SC - subcutaneous route of administration.

[0068] 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 performed using the GUSAR Online Acute Toxicity web service. This service predicted LD50 values ​​in rats for various routes of administration (intraperitoneal, intravenous, oral, and subcutaneous routes of administration). Table 3 also presents the results of predicting LD50 values ​​for compounds with predicted activity in inhibiting chemokine receptors.

[0069] From the data presented in Table 3 it is evident that the studied compounds are non-toxic or low-toxic compounds.

[0070] The binding of compounds to biological targets was also assessed using molecular docking.

[0071] Molecular docking is based on the assessment of ligand binding to the receptor region and provides information on its structural features, including conformations, orientation, and organization at the receptor site [L. Ferreira, R. dos Santos, G. Oliva, and A. Andricopulo, “Molecular docking and structure-based drug design strategies,” Molecules, vol. 20, no. 7, pp. 13384–13421, 2015]. Chemokine receptors are a type of GPCR receptor. Receptors of this group activate cellular responses through interactions with cognate G proteins. Similar interactions have been shown to be observed for the Vercimon ligand in the CCR9 chemokine receptor. 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 Tyr317. These amino acids play a key role in the formation of the allosteric binding site of CCR9.Compound la was considered as an antagonist for the orthosteric binding site in the chemokine receptors CCR7 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 - l- l- and / or l- cation-stacking interactions similar to those of the reference ligands. All studied compounds were subjected to the molecular docking procedure in the binding sites of CCR7 and CCR9.

[0072] Preparation of ligands and receptor

[0073] Crystallographic structures of the CCR7 receptor in complex with the antagonists Cmp2105 (PDB code 6QZH) and the CCR9 receptor in complex with Vercirnon (PDB code 5LWE) for modeling the binding process were downloaded from the non-commercial Protein Data Bank. Geometrical parameters of each receptor were prepared for calculations as follows: hydrogen atoms were added and minimized, water molecules and other low-molecular structures, except for the reference compounds, were removed, the whole structure was limitedly optimized by the OPLS3e force field method at physiological pH values ​​of the medium.

[0074] The geometric parameters of potential ligands were collected into a single database and optimized taking into account all acceptable conformations.

[0075] Binding site analysis

[0076] 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. Thr93, Arg94, Val331, Lys332 and Phe333 can be considered as functional amino acids for binding antagonists - with which the studied antagonist Cmp2105 forms hydrogen bridges for CCR7.

[0077] For CCR9, Arg323, Phe324 - hydrogen bonds, Argl44 and Arg323 - salt bridges, Tyr317 - n-n-stacking interactions between the aromatic ring of the amino acid and the chlorophenyl and phenyl rings of the ligand can be considered. The amino acid numbering corresponds to PDB codes 6QZH and 5LWE.

[0078] Molecular docking

[0079] 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 (IFD) protocol with the following conditions: flexible protein and ligand, grid matrix size of 15 Å, amino acids within 5 Å of the ligand are constrainedly optimized taking into account the ligand influence. The ranking of the docking solutions was performed by evaluating the following computational parameters: docking score (based on GlideScore minus penalties), ligand efficiency (LE, which takes into account the per-atom distribution of the scoring function), and the model energy value parameter (Emodel), 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.

[0080] Results of the assessment of binding of compounds to biological targets.

[0081] The results of molecular docking into the above-described binding sites of the antagonist and CCR7 receptor for the lead compounds are presented in Table 4.

[0082] Table 4. Molecular docking results of CCR7 receptor binding

[0083]

[0084] Compound 1a showed high binding in both CCR7 and CCR9 receptor. Notably, compound 1a binds in both sites. Compound 1a forms similar interactions in both sites: hydrogen bridges and π-π'-stacking interaction with the aromatic ring of tyrosine. In CCR7, compound 1a forms a hydrogen bridge between the oxygen atom and the amino group of arginine, and in CCR9, 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.The interaction with Argl54 and Tyr326, located in the cytoplasmic cleft part (opened upon GPCR activation) 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 Cmp2105 and Vercimon.

[0085] Example 3

[0086] Evaluation 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 tables below.

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] As follows from the experimental results, all compounds have similar inhibitory activity.

[0107] Example 5

[0108] Obtaining a pharmaceutical composition

[0109] Compound crystals

[0110] By weight - 2.5 mt, mixed with lactose (0.3 mt), mannitol (0.3 mt) and placed in a blender to obtain a homogeneous mixture. Then, the resulting mixture was moistened with a solution containing water, glycerin (2%), and transferred to a wet granulator. Then, in the dryer-granulator, granulation is carried out in a fluidized bed of pre-mixed component particles at a temperature of 40-80 °C, a feed rate of the granulating binder solution of 60-300 g / min and a binder solution concentration of 10% and granulate is calibrated on a vertical cone calibrator under the following modes: rotor frequency 80-600 rpm, gap between the grid and the rotor 1-3 mm, grid diameter 0.45-2 mm. An aqueous solution of povidone with a concentration of 10% was used as a binder by dissolving 0.50 kg of povidone in 5.0 kg of purified water. The resulting wet granules of powdered substances were dried for 5-10 min and calibrated on a vertical cone calibrator at a rotor speed of 100-120 rpm.0.230 g of magnesium stearate was added to the resulting granules. The resulting mixture was then tableted on a rotary press. Biconvex tablets were obtained that corresponded to the main hardness and dissolution parameters given for uncoated tablets.

Claims

Invention formula 1. Compounds selected from the following:

2. Compounds according to claim 1 for use as an inhibitor of the chemokine receptor CCR7.

3. Compounds according to paragraphs 1-2, characterized in that they are the compound N-(1,3-dioxoisoindol-5-yl)-2-naphthalen-1-ylacetamide 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 item 4, containing from 0.1 nM to 10 nM of the compound according to item 1.

7. Use of a compound according to paragraphs 1-3 in a therapeutically effective amount for the treatment of breast, esophageal, and lung cancer.

8. Use of compound 1-3 as an inhibitor of the chemokine receptor CCR7.

9. The use according to claim 7, characterized in that the chemokine receptor is the CCL21 receptor.

Images