Guanidines: Synthesis of Novel Histamine H3R Antagonists with Additional Breast Anticancer Activity and Cholinesterases Inhibitory Effect

Abstract

This study examines the properties of novel guanidines, designed and synthesized as histamine H₃R antagonists/inverse agonists with additional pharmacological targets. We evaluated their potential against two targets viz., inhibition of MDA-MB-231, and MCF-7 breast cancer cells viability and inhibition of AChE/BuChE. ADS10310 showed micromolar cytotoxicity against breast cancer cells, combined with nanomolar affinity at hH₃R, and may represent a promising target for the development of an alternative method of cancer therapy. Some of the newly synthesized compounds showed moderate inhibition of BuChE in the single-digit micromolar concentration ranges. H₃R antagonist with additional AChE/BuChE inhibitory effect might improve cognitive functions in Alzheimer's disease. For ADS10310, several in vitro ADME-Tox parameters were evaluated and indicated that it is a metabolically stable compound with weak hepatotoxic activity and can be accepted for further studies.

Keywords: antagonist; breast cancer; guanidines; histamine H3 receptor; multi-target directed ligand.

Figure 1

Example structures of non-imidazole H₃R antagonists.

Scheme 1

The target molecules of this study.

Scheme 2

Synthesis of ADS10377, ADS10376. Reagents and conditions: (a) 1,7-dibromoheptane (1 equiv.), 4-chlorophenol (1 equiv.), sodium phenoxide (1 equiv.), EtOH, 24 h, 80 °C; (b) 1 (1 equiv.), piperazine (5 equiv.), MeOH, 24 h, 70 °C; (c) 2 (1 equiv.), 4-bromobutyronitrile (1.3 equiv.), potassium carbonate (5 equiv.), MeCN, 24 h, 80 °C; (d) 3 (1 equiv.), LiAlH₄ (4 equiv.), diethyl ether, 24 h, rt; (e) 4 (1 equiv.), benzoyl chloride (1.1 equiv.) or 4-(trifluoromethyl)benzoyl chloride (1.1 equiv), triethylamine (5 equiv.), DCM, 3 h, rt; (f) 5a/5b (1 equiv.), LiAlH₄ (4 equiv.), diethyl ether, 24 h, rt; (g) 6a/6b (1 equiv.), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv.), HgCl₂ (1.1 equiv.), triethylamine (5 equiv.), DCM, 18 h, rt; (h) 7a/7b (1 equiv.), 4M solution HCl-dioxan (20 equiv.), CHCl₃, 24h, rt.

Scheme 3

Synthesis of (3-bromopropyl)benzene (A), 3-(4-chlorophenyl)propyl methanesulfonate (B), and ADS10349, ADS10350, ADS10278, ADS10279 (C). Reagents and conditions: (a) 3-Phenyl-1-propanol (1 equiv.), phosphorus tribromide (1.4 equiv.), toluene, 24 h, rt; (b) 3-(4-Chlorophenyl)-1-propanol (1 equiv.), methanesulfonyl chloride (1.4 equiv.), triethylamine (1.9 equiv.), DCM, 1 h, rt; (c) 4-bromobutyronitrile (1 equiv.), piperazine (5 equiv.), CHCl₃, 2 h, 60 °C; (d) 10 (1equiv.), 3-Bromo-1-propanol (1.08 equiv.), triethylamine (4 equiv.), MeCN, 24 h, 80 °C; (e) 11 (1 equiv.), 8 (1 equiv.), TBAI (0.05 equiv.), 40% NaOH, 24 h, rt; (e) 11 (1 equiv.), 9 (1.2 equiv.), sodium hydride (60% dispersion in mineral oil) (1.6 equiv.), N,N-dimethylacetamide, 24 h, rt; (f) 12a/12b (1 equiv.), LiAlH₄ (4 equiv.), diethyl ether, 24 h, rt; (g) 13a/13b (1 equiv.), benzoyl chloride (1.1 equiv.) or 4-(trifluoromethyl)benzoyl chloride (1.1 equiv), triethylamine (5 equiv.), DCM, 3 h, rt; (h) 14a/14b/14c/14d (1 equiv.), LiAlH₄ (4 equiv.), diethyl ether, 24 h, rt; (i) 15a/15b/15c/15d (1 equiv.), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv.), HgCl₂ (1.1 equiv.), triethylamine (5 equiv.), DCM, 18 h, rt; (j) 16a/16b/16c/16d (1 equiv.), 4M solution HCl-dioxan (20 equiv.), CHCl₃, 24 h, rt.

Scheme 4

Synthesis of semi-products: 3-(piperidin-1-yl)propan-1-amine and 4-(piperidin-1-yl)butan-1-amine (A), 1-(3-bromopropyl)-4-chlorobenzene (B), and 1-(4-bromobutyl)-4-chlorobenzene (C). Reagents and conditions: (a) Piperidine (1 equiv.), Acrylonitrile (1.1 equiv.), MeOH, 24 h, rt or Piperidine (1 equiv.), 4-bromobutyronitrile (1 equiv.), potassium carbonate (5 equiv.), MeCN, 72 h, rt; (b) 17a/17b (1 equiv.), LiAlH₄ (4 equiv.), diethyl ether, 24 h, rt; (c) 3-(4-Chlorophenyl)-1-propanol (1 equiv.), phosphorus tribromide (1.2 equiv.), toluene, 24 h, rt; (d) 3-(4-chlorobenzoyl)propionic acid (1 equiv.), Borane-tert-butylamine-complex (6 equiv.), aluminum chloride (4 equiv.), 216 h, 40 °C; (e) 20 (1 equiv.), phosphorus tribromide (1.4 equiv.), toluene, 24 h, rt.

Scheme 5

Synthesis of ADS10292, ADS10300, ADS10312. Reagents and conditions: (a) 18a/18b (1 equiv.), 19/21 (1 equiv.), potassium carbonate (5 equiv.), MeCN, 24 h, 60 °C; (b) 22a/22b/22c (1 equiv.), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv.), HgCl₂ (1.1 equiv.), triethylamine (5 equiv.), DCM, 18 h, rt; (c) 23a/23b/23c (1 equiv.), 4M solution HCl-dioxan (20 equiv.), CHCl₃, 24 h, rt.

Scheme 6

Synthesis of ADS10298, ADS10301, ADS10306, ADS10310. Reagents and conditions: (a) 3-(4-Chlorophenyl)-1-propanol or 20 (1 equiv.), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (0.5 equiv.), triphenylphosphine (1.1 equiv.), 94% DIAD (1.1 equiv.), THF, 24 h, rt; (b) 24a/24b (1 equiv.), 18a/18b (2 equiv.), water, THF, 24 h, 70 °C; (c) 25a/25b/25c/25d (1 equiv.), 4M solution HCl-dioxan (20 equiv.), CHCl₃, 24 h, rt.

Figure 2

The relative viability of cells treated with ADS10310 (a), ADS1017 (b) or DMSO as the vehicle in control culture for 48 h (determined by MTT test). The results were calculated as % of control culture viabilities which were averaged to define the 100%. Values were presented as mean ± SD derived from three independent experiments. * p < 0.05 vs. control. BJ—skin fibroblasts.

Figure 3

MDA-MB-231 and MCF-7 breast cancer cells treated with ADS10310 and ADS1017 in concentrations corresponding to IC₅₀ values for 48 h (control cultures treated with DMSO as the vehicle). (a) Morphology. Magnification ×200. (b) Apoptosis/necrosis detection by image cytometry. The results show one representative experiment of three independently performed. Q1II—live, Q1Ir—early apoptotic, Q1ur—late apoptotic, and Q1uI—necrotic cells. (c) Cell cycle analysis results based on DAPI staining and image cytometry measurement. The results show one representative experiment of three independently performed M1—subG1, M2—G1, M3—S, M4—G2/M phase.

Figure 4

Binding mode of compound ADS10310 within H₃R.

Figure 5

UPLC spectra after 120 min incubation of compound ADS10310 in TRIS buffer pH = 7.4 at 37 °C with HLMs. A total of 73.63% of the parent compound remained in the reaction mixture.

Figure 6

The influence of ADS10310 and the reference inhibitor: (a) ketoconazole (KE, 1 µM) on CYP3A4 activity (b) quinidine (QD, 1 µM) on CYP2D6 activity. (c) The effect of ADS10310 and cytostatic drug doxorubicin (DOX, 1 µM) on hepatoma HepG2 cell line viability after 72 h of incubation at 37 °C, 5% CO₂. Statistical significance (**** p < 0.0001) was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s Multiple Comparison.