Next Article in Journal
A Rapid and Reliable Spectrofluorimetric Method to Measure the Urinary Lactulose/Mannitol Ratio for Dysbiosis Assessment
Previous Article in Journal
Evaluation of Neuroinflammatory Contribution to Neurodegeneration in LRRK2 Drosophila Models
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biomedicines
Volume 12
Issue 7
10.3390/biomedicines12071556
biomedicines-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Spasmolytic Activity of 1,3-Disubstituted 3,4-Dihydroisoquinolines

by
Miglena Milusheva
1,2,
Mihaela Stoyanova
1,
Vera Gledacheva
3,
Iliyana Stefanova
3,
Mina Todorova
1 and
Stoyanka Nikolova
1,*
1
Department of Organic Chemistry, Faculty of Chemistry, University of Plovdiv, 4000 Plovdiv, Bulgaria
2
Department of Bioorganic Chemistry, Faculty of Pharmacy, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria
3
Department of Medical Physics and Biophysics, Faculty of Pharmacy, Medical University of Plovdiv, 4002 Plovdiv, Bulgaria
*
Author to whom correspondence should be addressed.
Biomedicines 2024, 12(7), 1556; https://doi.org/10.3390/biomedicines12071556
Submission received: 9 June 2024 / Revised: 2 July 2024 / Accepted: 11 July 2024 / Published: 13 July 2024
(This article belongs to the Special Issue Advances in Angiogenesis, Inflammation, and Oxidative Stress in Inflammatory Bowel Diseases)
Browse Figures
Versions Notes

Abstract

:
This article concerns the spasmolytic activities of some novel 1,3-disubstituted 3,4-dihydroisoquinolines. These compounds can be evaluated as potential therapeutic candidates according to Lipinski’s rule of five, showing high gastrointestinal absorption and the ability to cross the blood–brain barrier, which is a very important parameter in the drug discovery processes. In silico simulation predicted smooth muscle relaxant activity for all the compounds. Since smooth muscle contractile failure is a characteristic feature of many disorders, in the current paper, we concentrate on the parameters of the spontaneous contractile responses of smooth muscle (SM) cells compared to the well-known drug mebeverine. Two of the newly synthesized substances can be identified as essential modulating regulators and potentially used as therapeutic molecules. One of these molecules also showed significant DPPH antioxidant activity compared to rutin.

1. Introduction

The isoquinolines are a family of phytochemicals found in plants, like Papaveraceae, Berberidaceae, and Ranunculaceae. The alkaloids found in these plants possess a remarkable number of biological activities. The isoquinoline ring has been found to possess a wide range of biological and pharmacological activities, for example, antimalarial, anti-HIV, insect-growth-retarding, antitumor, antimicrobial, antifungal, antiparasitic and insecticidal, antiviral, anti-inflammatory, and antiplatelet, activities [1,2,3]. The first isolated and reported isoquinoline alkaloid from Papaver somniferum at the beginning of the 19th century was morphine. More than 2500 new structures have since been described, turning the isoquinolines into one of the most complex and diverse groups of alkaloids [4,5]. Due to their vast chemical versatility, isoquinolines have been grouped into 13 major subclasses, such as bisbenzylisoquinolines, pavines, cularines, aporphines, promorphinans, protoberberines, berberines, and simple benzylisoquinolines [6]. Papaverine, for example, belongs to the benzylisoquinoline alkaloids. For decades, this compound has been known as a brain and coronary vasodilator and muscle relaxant, and for its non-specific spasmolytic activity [7]. Papaverine has been known for ages because of its spasmolytic activity [8,9,10,11,12,13], but it found applications in the inhibition of mitochondrial metabolism, the enhancement in tumor oxygenation [14,15], HCoV–OC43 treatment [16], etc. Therefore, an isoquinoline can be an important starting point for drug discovery. The wide range of biological activities associated with isoquinolines set our interest in obtaining novel derivatives and establishing a correlation between their structure and activity.
In the search for novel biologically active molecules, we present here a simple synthesis of 1,3-disubstituted 3,4-dihydroisoquinolines. Using previously described amides [17], the current work attempted to synthesize a number of 3,4-dihydroisoquinoline analogs via the Bischler–Napieralski reaction.
To further understand their biological characteristics, all of these substances were evaluated for their potential spasmolytic activity. Antispasmodics are typically used to reduce excessive smooth muscle spasms, which can be caused by a variety of illnesses and result in stomach cramps and pain. The genitourinary, biliary, or gastrointestinal tract is affected by these spasms. Antispasmodics are used to treat biliary colic and irritable bowel syndrome (IBS) by decreasing the contractility of smooth muscle [18].

2. Materials and Methods

All solvents and reagents were purchased from Merck (Merck KgaA, Darmstadt, Germany). The compounds were characterized by their 1H-NMR, 13C-NMR, and HRESIMS spectra. The purity of the compounds was determined by TLC (precoated 0.2 mm Merck silica gel 60 plates (Merck KgaA, Darmstadt, Germany)). NMR spectra were recorded at room temperature (ca. 295 K) on a Bruker Avance III HD 500 spectrometer (Bruker, Billerica, MA, USA) at 500 MHz.
HRESIMS spectra were acquired in positive mode on a Q Exactive Plus (ThermoFisher Scientific, Inc., Bremen, Germany) mass spectrometer equipped with a heated HESI-II source. Operating conditions for the HESI source used in positive ionization mode were +3.5 kV spray voltage, 320 °C capillary and probe heater temperature, sheath gas flow rate of 36 a.u., auxiliary gas flow rate of 11 a.u., spare gas flow rate of 1 a.u. (a.u. refers to the arbitrary values set by Exactive Tune software 2.4), and S-Lens RF level of 50.00. Nitrogen was used for sample nebulization and as collision gas in the HCD cell. Aliquots of 1 µL of the solutions of the samples (ca. 20 µg mL−1) were introduced into the mass spectrometer through the LC system, Thermo Scientific Dionex Ultimate 3000 RSLC (Germering, Germany), consisting of 6-channel degasser SRD-3600, high-pressure gradient pump HPG-3400RS, autosampler WPS-3000TRS, and column compartment TCC-3000RS equipped with narrow-bore Hypersil GOLD™ C18 (2.1 × 50 mm, 1.9 μm) column. Each chromatographic run was carried out isocratically with a mobile phase consisting of water–acetonitrile–methanol–acetic acid (25:50:25:0.2). The solvent flow rate was 300 μL min−1. Full MS–SIM was used as MS experiment in negative and positive mode, where the resolution, automatic gain control (AGC) target, maximum injection time (IT), and mass range were 70,000 (at m/z 200), 3 × 106, 100 ms, and m/z 100–500, respectively. Xcalibur (Thermo Fisher Scientific, Waltham, MA, USA) ver. 4.0 was used for data acquisition and processing.

2.1. Experimental Protocols and Spectral Data

To a solution of 3 mmol of the corresponding amide 4 [17] in 1,2-dichloroethane, 1 mL of phosphorus(V) oxychloride was added. The reaction mixture was refluxed for 1 h at 100 °C, poured in water, then extracted with CH2Cl2 (3 × 20 mL), and washed with Na2CO3 and water. The organic layer was dried using anhydrous Na2SO4, filtered on the short column filled with basic Al2O3, and then concentrated.
3-isopropyl-1-methyl-3,4-dihydroisoquinoline (5a), yellow oil, 62% yield, 1H-NMR: 7.26–7.29 (m, 2H), 7.19–7.22 (m, 3H), 4.07 (ddd, 1H, J = 9.3, 5.6, 3.7, CH), 2.88 (dd, 1H, J = 13.7, 5.4, CH2), 2.74 (dd, 1H, J = 14.2, 8.8, CH2), 1.9 (s, 3H, COCH3), 1.82–1.86 (m, 1H, CH(CH3)2), 1.0 (d, 3H, J = 6.8, CH(CH3)2), 0.98 (d, 3H, J = 6.8, CH(CH3)2); 13C-NMR: 171.02, 138.42, 129.16, 128.36, 126.37, 56.46, 37.88, 30.99, 22.37, 19.58, 17.92; HRMS Electrospray ionization (ESI) m/z calcd. for [M+H]+ C13H17N+ = 188.28078, found 188.27036 (mass error ∆m = −1.04 ppm).
3-isopropyl-1-phenyl-3,4-dihydroisoquinoline (5b), yellow oil, 70% yield, 1H-NMR: 7.61–7.63 (m, 2H), 7.45–7.47 (m, 1H), 7.36–7.40 (m, 2H), 7.18–7.26 (m, 5H), 4.30–4.35 (m, 1H, CH), 2.93–2.97 (m, 1H, CH2), 2.81–2.85 (m, 1H, CH2), 1.85–1.92 (m, 1H, CH(CH3)2), 1.03 (d, 3H, J = 6.4, CH(CH3)2), 1.01 (d, 3H, J = 6.4, CH(CH3)2); 13C-NMR: 167.28, 138.32, 135.12, 131.23, 129.22, 128.53, 128.47, 126.41, 55.51, 37.98, 30.77, 19.70, 17.84.; HRMS Electrospray ionization (ESI) m/z calcd. for [M+H]+ C18H20N+ = 250.15903, found 250.15866 (mass error ∆m = −1.46 ppm).
1-(2-chlorophenyl)-3-isopropyl-3,4-dihydroisoquinoline (5c), yellow oil, 75% yield, 1H-NMR: 7.45–7.47 (m, 1H), 7.35–7.36 (m, 1H), 7.29–7.33 (m, 3H), 7.24–7.26 (m, 3H), 4.34–4.42 (m, 1H, CH), 2.96 (dd, 1H, J = 14.2, 6.4, CH2), 2.79 (dd, 1H, J = 14.2, 8.3, CH2), 1.88–1.94 (m, 1H, CH(CH3)2), 1.06 (d, 3H, J = 6.5, CH3), 1.02 (d, 3H, J = 7.0, CH3); 13C-NMR: 166.43, 138.29, 135.58, 133.21, 132.24, 131.01, 130.06, 129.87, 126.98, 126.64, 126.41, 55.95, 38.18, 30.84, 19.73, 17.55; HRMS electrospray ionization (ESI) m/z calcd. for [M+H]+ C18H19NCl+ = 284.12005, found 284.11966 (mass error ∆m = −1.39 ppm).
1-benzyl-3-isopropyl-3,4-dihydroisoquinoline (5d), yellow oil, 72% yield, 1H-NMR: 7.28–7.33 (m, 3H), 7.18–7.23 (m, 2H), 7.09–7.11 (m, 2H), 7.02–7.03 (m, 2H), 4.05–4.10 (m, 1H, CH), 3.47 (q, 2H, J = 16.1, CH2), 2.76 (dd, 1H, J = 14.2, 5.9, CH2), 2.51 (dd, 1H, J = 14.2, 8.3, CH2), 1.65–1.72 (m, CH(CH3)2, 1H), 0.89 (d, 3H, CH(CH3)2, J = 6.4), 0.79 (d, 3H, CH(CH3)2, J = 6.8); 13C-NMR: 170.35, 138.20, 134.95, 129.47, 129.14, 128.35, 127.31, 126.28, 55.05, 43.99, 37.81, 30.77, 19.43, 17.57; HRMS electrospray ionization (ESI) m/z calcd. for [M+H]+ C19H22N+ = 264.17468, found 264.17433 (mass error ∆m = −1.31 ppm).

2.2. In Silico Pharmacokinetic Profiling and Toxicity Analysis

2.2.1. Theoretical Prediction of Pharmacokinetic Parameters—Absorption, Distribution, Metabolism, and Excretion (ADME) Properties

Physicochemical and pharmacokinetic parameters, and drug-likeness of SQ were analyzed using SwissADME, a free web tool [19].

2.2.2. Theoretical Prediction of Toxicity

The ProToxII web tool was used to predict acute and organ toxicity, toxicity class, and LD50 for the compound [20,21].

2.2.3. PASS Online Predictions

A computer-based PASS online program was used to predict biological activities of the compound, based on its structural formula [22,23,24,25].

2.3. Smooth Muscle Activity

2.3.1. Ex Vivo Experiments on Gastric Smooth Muscle Preparations (SMPs) from Wistar Rats

Wistar rats (270 ± 15 g body weight, male, 11 weeks of age) were purchased from the Medical University Plovdiv, Animal Laboratory, Bulgaria. The experimental procedures were conducted with the highest confidence, adhering to the Guidelines for the Care and Use of Laboratory Animals, Medical University of Plovdiv, Bulgaria, permit No. 229/09 April 2019. The procedures were approved by the current European regulations (86/609/EEC), complied with EU Directive 2010/63/EU, and performed in strict accordance with the current guidelines of Institutional Animal Care Bulgaria.
Four circular pieces (9–11 mm in length, 1.0–1.2 mm in width) of muscle tissue were taken from one rat stomach, with the mucosa remaining intact. As indicated by n, the samples were acquired under the following conditions: 4 °C temperature and continuous irrigation of tissues with pre-aerated (95% O2 and 5% CO2) preparation solution containing NaCl/KCl/CaCl2 in a ratio of 27.2/1.1/1 [25]. The samples were equilibrated in a typical physiological salt Krebs solution containing 140.0 mM NaCl, 5.0 mM KCl, 1.2 mM MgCl2, 1.8 mM CaCl2, 23.8 mM NaHCO3, and 11.1 mM glucose at a resting tension of 10 mN (1 g). Before each experiment, the pH of the solution was determined using an HI5521 pH meter (Hanna Instruments, Smithfield, RI, USA). The bath solution was saturated at 37 °C, pH 7.4, with a mixture of 95% O2 and 5% CO2.

2.3.2. Method for Studying the Mechanical Activity of Isolated SMPs

Experiments were performed to investigate the cumulative dose-dependent relaxation effects of compounds 5a–d on gastric SMPs, pre-contracted with CCh (1 × 10−7 mol/L) using the previously described method [25].

2.4. Ethics Statement

The experiments were approved by the Ethical Committee of the Bulgarian Food Agency with permit No. 229/09.04.2019 and were carried out following the guidelines of European Directive 2010/63/EU. The animals were provided by the Medical University-Plovdiv (Animal House, Plovdiv, Bulgaria).

2.5. DPPH Free Radical Scavenging Assay

The DPPH free radical scavenging activity was evaluated by applying Docheva‘s method [26].

2.6. Statistical Analysis

For the analysis of variance, the InStat v. 3.10 (GraphPad Software, Inc., San Diego, CA, USA) computer program was utilized. Each group’s mean and standard error of the mean were determined. To compare various groups with their corresponding controls for repeated measurements, a two-way ANOVA was employed. A significant difference was deemed to be represented by a p-value of less than 0.05. The statistical software IBM SPSS Statistics v. 26 was utilized for the analyses.

3. Results and Discussion

Abdominal pain and difficulty voiding are two of the complex symptoms of IBS. For many years, the anticholinergic spasmolytic mebeverine has been used to treat IBS. In addition, it functions as a musculotropic drug, calcium channel blocker, and an antispasmodic, and it has regulatory effects on bowel function [27]. Mebeverine, as a second-generation papaverine analog (Figure 1), reduces the excessive contractility of smooth muscle (SM) cells, giving it direct myolytic activity.
Finding novel therapeutic strategies to influence established pharmacological targets or validate novel compounds with an alternate mode of action for IBS remains a top objective. Recently, we described the synthesis of 1-(3,4-dimethoxyphenyl)propan-2-amides and 3-methyl-1-phenylbutan-2-amides, their SM relaxant, and bioelectrical activities, and their influence on cognitive functions [17,28]. In the current work, we considered the synthesis of 1,3-disubstituted 3,4-dihydroisoquinolines due to their structural similarity to mebeverine and papaverine and their pharmacological properties as antispasmodics.

3.1. Synthetic Method

Our strategy is based on the synthesis of 1-(3,4-dimethoxyphenyl)propan-2-amine and 3-methyl-1-phenylbutan-2-amine [17,28] from a starting ketone and its acylation with acid halides to corresponding amides.
Next, we turned our attention toward the incorporation of acyl residue into N-heterocycles using the Bischler–Napieralski reaction to generate the isoquinoline core (Scheme 1). We found that the application of amide 4 in the Bischler–Napieralski reaction with phosphorus(V)oxychloride in 1,2-dichloroethane and a reflux for 1–3 h yields the corresponding 1-substituted 3-isopropyl products 5a–d in moderate yields. The NMR data confirmed the structure of the obtained compounds (Supplementary Materials Figures S1–S8).

3.2. In Silico Predictions

Using in silico methods, the drug-likeness of the compounds and their ADMET properties have also been predicted theoretically. Increased lipophilicity, membrane permeability, and pharmacological activity are the outcomes of the conformational restriction of small drug molecules brought about by the incorporation of this noncovalent interaction into drug design [29].
According to Lipinski’s rule of five [30], the ADME investigation demonstrates that the compounds have good gastrointestinal absorption and can pass across the blood–brain barrier. We found that 5a–d met the standard values (Table 1) of molecular weight (MW ≤ 500), XLOGP3 ≤ 5, ESOL or estimated solubility (logS: not more than six), saturation (Fraction Csp3 or fraction of carbons in the sp3 hybridization: not less than 0.25), and flexibility (RB: no more than nine).
A Csp3 of 0.28–0.46 and 1–3 rotatable bonds satisfied the standard value of the drug-likeness concept. The compounds scored a bioavailability 0.55, which relates to good oral absorption [31]. They also had a good synthetic accessibility score (3.12–3.63), which is a very important parameter in the drug discovery process. Pharmacological toxicity is a consequence of cytochrome P450 (CYP) inhibition [32,33]. The studied compounds are not predicted as P-glycoprotein substrates. The calculations showed that 5b and 5d are expected to inhibit the CYP1A2, CYP2D6, and CYP3A4 isoforms, while 5a and 5c are expected to inhibit the CYP2D6 and CYP3A4 isoforms. The compounds have a log Kp between −4.28 and −5.31, which indicate good skin permeability.
Acute toxicity data (Pro Tox-II) indicated that the toxicity class of all the compounds is class 3. The LD50 predicted for all the compounds is 240 mg/kg. 5b and 5d were predicted to have mutagenicity (0.55% probability), whereas other organ toxicities were absent.
As a result, all the synthesized compounds can be evaluated as potential therapeutic candidates. The PASS Online Program predicted the muscle relaxant and antioxidant activities of the synthesized compounds. Once synthesized, we turned our attention to investigating the biological activities of the synthesized 3,4-dihydroisoquinolines.

3.3. Spasmolytic Activity

Traditionally, smooth, skeletal, and cardiac muscles have been categorized as excitable tissues with phenotypic variety, capable of contracting or relaxing in response to endogenous or external pharmacological agents. Since smooth muscle contractile failure is a characteristic feature of many disorders, in the current paper, we investigated the parameters of the spontaneous contractile responses of smooth muscle cells.
There are two types of smooth muscle: tonic and phasic. Vascular dilatation occurs in tonic smooth muscles. The gastrointestinal and urogenital systems are home to phasic smooth muscles, which, as their name implies, contract rhythmically. The frequency, amplitude, and strength of the tonic response define the phasic contraction of phasic smooth muscles, which operate as if they are solitary muscles. Interstitial cells of Cajal, which are pacemaker cells, can start phasic contractions, and they can be sped up or slowed down by turning on an electromechanical or pharmacomechanical coupling. These processes can be modulated by muscle-specific signals depending on the type of muscle, its function, and the amount of force required. The force in pharmacomechanical coupling is triggered by receptor signaling, which may or may not entail a change in the membrane potential. In electromechanical coupling, the force is triggered by a change in the membrane potential [34].
This distinction is important when examining the effects of freshly produced pharmacological drugs or their precursors and analogs on smooth muscle function ex vivo, as all smooth muscles can be stimulated by both methods. Early research on smooth muscle in animals (dogs and rabbits) led to the distinction between pharmacomechanical coupling, which produces action potentials and phasic contractile activity, and electromechanical coupling, which results in graded depolarizations and contractions [35].
It is currently understood that vasoconstrictor signals cause the smooth muscle contraction through two mechanisms: (1) calcium influx, which activates myosin light chain kinase (MLCK); and (2) second messengers, which inhibit the myenteric plexus (MP) and make myofilaments more sensitive to calcium activation [36]. These mechanisms are active at cytosolic Ca2+ levels of the order of 10−5 M, according to Droogmans and Casteels [37]. Vasodilators, on the other hand, relax smooth muscle by desensitizing myofilaments to calcium by blocking calcium influx and activating the MP. Mebeverine is a well-known medication with direct musculotropic activity that involves Ca2+ ion exchange and excitable membrane stability [38]. It is a well-known drug that has been shown to help with irritable bowel syndrome, but it also has a lot of unfavorable side effects [39].
Thus, it makes sense and supports our scientific purpose to compare the myotropic effect of mebeverine with recently reported compounds that have a comparable structure through a functional analysis of their activating and inhibiting abilities. In a prior investigation, we used the single sucrose gap approach to determine these bioelectrical effects for the isoquinoline precursors [17]. In the present work, we compared the spasmolytic activity of 3,4-dihydroisoquinolines 5a–d to their previously described precursors 4a–d.
Antispasmodics can be categorized into two groups based on their mechanism of action: neurotropic drugs, which block nerve impulses from reaching smooth muscle cells and alter muscle bioelectric activity; and myotropic drugs, which have an impact on the biochemical processes that control smooth muscle contractions.
The myotropic action in all muscle cells is expressed as contraction or relaxation and is dependent on changes in the concentration of cytosolic calcium [40].
SMPs from the central region of the rat stomach were studied in order to ascertain the biological effects of compounds 4a–d and 5a–d on the mechanical contractile activity of smooth muscle and to assess the effects of induced relaxation or contraction (Figure 2, Table 2). As a control, the response of SMPs to mebeverine was observed. This decision was influenced by the fact that the characteristics of the spontaneous contractile activity (SCA) of smooth muscle strips were unaffected by mebeverine for gastrointestinal disorders when it was supplied in the tissue bath at a concentration of 5 × 10–5 M.
Time efficiency was measured by observing all mechanical changes for 60 min under identical experimental conditions, with the investigated chemicals acting at a concentration of 5 × 10−5 M. We can classify substances 4a–d, 5a, and 5c as fast-acting as they reached their maximal biological potential within 4 min. Since 5b and 5d only became fully effective after 10 min, they can be classified as having a longer delayed action. The tonus component, frequency, and amplitude of contraction for 5b (Figure 3), as well as the tone and frequency of contraction, were reliably reduced in the isometric registration of the evoked smooth muscle responses [40]. However, the amplitude of the single contractions under impact with 5a and 5c remained unaffected. A relaxation effect was found for 4d; however, its tone and amplitude statistically changed (Table 2).
The SMPs completely relaxed and the contractions stopped in the background of compound 4c, designating it as a prototype SM relaxant. The induced relaxations can be attributed to the stimulation of calcium uptake, the inhibition of calcium influx, a direct decrease in intracellular Ca2+ concentration, or the inhibition of calcium release from intracellular storage sites [41]. The current paper aimed to determine the mechanism of these effects in the isolated intestinal SMPs.
Contrary to the mechanisms of the relaxation with substances 4b, 4d, 5b, 5a, and 5c, substances 4a and 4c did not exhibit any visible effect on the mechanical properties and contraction force of the SMPs, even at higher concentrations (up to 10−4 M), as determined by the limitations of the applied SM model in the ex vivo study.
The reported stimulating impact of compound 4d was unexpected given the software estimates and relaxation effects we confirmed for some of the tested compounds. Sharp, strong, and consistent in both amplitude and tone, the contraction likely impacts the activation of calcium-delivering contractile proteins or increases the flow of Ca2+ into the intracellular space (Table 2 and Figure 3).
Finally, signal transduction is essential for contractile cell function. We found that compound 5b demonstrates considerable relaxation by altering the primary parameters of tone and frequency of the SCA of gastric SMs. Substance 5d, in its capacity as a stimulating signal, likely influences calcium translocation, which promotes tonic and rhythmic muscular activity. These data give us a reason to identify the newly synthesized substances, especially 5b and 5d, as essential modulating regulators and potentially use them as therapeutic molecules.

3.4. DPPH Radical Scavenging Activity

Finally, the in silico methods predicted antioxidant activity for the compounds, and we evaluated the antioxidant potential in vitro using a DPPH radical scavenging assay [26] using quercetin and rutin as standards. We found that only compound 5d showed significant antioxidant potential compared to the standards.
The obtained data showed that 5d at a concentration of 31 ± 0.25 µM exhibited a percentage inhibition of 55%, which was compared to the antioxidant activities of quercetin (31 µM with 75.9% inhibition), rutin (31 µM with 30% inhibition), and mebeverine (31 µM with 8% inhibition) (Figure 4). The results showed that 5d has better antioxidant activity than rutin and mebeverine, but lower than that of quercetin.

4. Conclusions

A number of novel 3-isopropyl 3,4-dihydroisoquinolines were synthesized as potential antispasmodics. All the synthetic compounds, in general, displayed favorable expected in silico profiles and were identified as potentially useful oral medications with low toxicity. Isolated tissues were used to demonstrate their ex vivo spasmolytic activity compared to those of previously described precursors. The results demonstrate that compound 5d can act as a contractile agent in its capacity as a stimulating signal, probably influencing calcium translocation, which promotes tonic and rhythmic muscular activity, and compound 5b exhibits significant relaxation by changing the primary parameters of tone and frequency of the SCA of gastric SMs. Compound 5d also shows very good antioxidant activity, better than mebeverine and rutin but lower than that of quercetin.
Thus, the newly synthesized compounds 5b and 5d were found to have favorable spasmolytic activity and could be fair and successful options for the long-term oral therapy of chronic IBS, according to the summarized results of the conducted trials. Therefore, it is necessary to conduct additional in vivo research in the future to explore the potential therapeutic uses of these 3,4-dihydroisoquinolines.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/biomedicines12071556/s1, Figure S1: 1H-NMR spectrum of compound 5a, Figure S2: 13C-NMR spectrum of compound 5a, Figure S3: 1H-NMR spectrum of compound 5b, Figure S4: 13C-NMR spectrum of compound 5b, Figure S5: 1H-NMR spectrum of compound 5c, Figure S6: 13C-NMR spectrum of compound 5c, Figure S7: 1H-NMR spectrum of compound 5d, Figure S8: 13C-NMR spectrum of compound 5d.

Author Contributions

Conceptualization, S.N.; methodology, M.M., M.S., V.G. and M.T.; investigation, M.M., S.N., M.S., V.G., I.S. and M.T.; writing—original draft preparation, S.N., I.S. and V.G.; writing—review and editing, S.N.; visualization, S.N., I.S. and V.G.; supervision, S.N.; project administration, S.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The procedures used in this study agreed with the European Communities Council Directive 2010/63/EU for animal experiments. The experimental procedures were conducted following national rules on animal experiments and were approved by the Bulgarian Food Safety Agency (No. 229/No. 145/09.04.2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Acknowledgments

This study is part of Scientific Project No KP-06-H73/11 of the National Fund for Scientific Research in Bulgaria, National Program for Basic Research Projects – 2023.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Shang, X.; Yang, C.; Morris-Natschke, S.L.; Li, J.; Yin, X.; Liu, Y.; Guo, X.; Peng, J.; Goto, M.; Zhang, J.; et al. Biologically active isoquinoline alkaloids covering 2014–2018. Med. Res. Rev. 2020, 40, 2212–2289. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, D.-T.; Qin, L.-L.; Jing, C.-X.; Wang, G.-H.; Zhou, H.; Deng, P.; Zhang, S.-Y.; Wang, Y.-R.; Ding, Y.-Y.; Zhang, Z.-J.; et al. Biologically active isoquinoline alkaloids covering 2019–2022. Bioorganic Chem. 2024, 145, 107252. [Google Scholar] [CrossRef] [PubMed]
  3. Dey, P.; Kundu, A.; Kumar, A.; Gupta, M.; Lee, B.M.; Bhakta, T.; Dash, S.; Kim, H.S. Analysis of alkaloids (indole alkaloids, isoquinoline alkaloids, tropane alkaloids). In Recent Advances in Natural Products Analysis; Elsevier: Amsterdam, The Netherlands, 2020; pp. 505–567. [Google Scholar]
  4. Gaber, A.; Alsanie, W.F.; Kumar, D.N.; Refat, M.S.; Saied, E.M. Novel Papaverine Metal Complexes with Potential Anticancer Activities. Molecules 2020, 25, 5447. [Google Scholar] [CrossRef] [PubMed]
  5. Gomes, D.A.; Joubert, A.M.; Visagie, M.H. The Biological Relevance of Papaverine in Cancer Cells. Cells 2022, 11, 3385. [Google Scholar] [CrossRef] [PubMed]
  6. Plazas, E.; Muñoz, D.R. Natural isoquinoline alkaloids: Pharmacological features and multi-target potential for complex diseases. Pharmacol. Res. 2022, 177, 106126. [Google Scholar] [CrossRef] [PubMed]
  7. Chulia, S.; Ivorra, M.D.; Martinez, S.; Elorriaga, M.; Valiente, M.; Noguera, M.A.; Lugnier, C.; Advenier, C.; D’Ocon, P. Relationships between structure and vascular activity in a series of benzylisoquinolines. Br. J. Pharmacol. 1997, 122, 409–416. [Google Scholar] [CrossRef] [PubMed]
  8. Ashrafi, S.; Alam, S.; Sultana, A.; Raj, A.; Emon, N.U.; Richi, F.T.; Sharmin, T.; Moon, M.; Park, M.N.; Kim, B. Papaverine: A Miraculous Alkaloid from Opium and Its Multimedicinal Application. Molecules 2023, 28, 3149. [Google Scholar] [CrossRef] [PubMed]
  9. Song, D.; Hao, J.; Fan, D. Biological properties and clinical applications of berberine. Front. Med. 2020, 14, 564–582. [Google Scholar] [CrossRef] [PubMed]
  10. Rauf, A.; Abu-Izneid, T.; Khalil, A.A.; Imran, M.; Shah, Z.A.; Emran, T.B.; Mitra, S.; Khan, Z.; Alhumaydhi, F.A.; Aljohani, A.S.M.; et al. Berberine as a Potential Anticancer Agent: A Comprehensive Review. Molecules 2021, 26, 7368. [Google Scholar] [CrossRef]
  11. Habtemariam, S. Berberine pharmacology and the gut microbiota: A hidden therapeutic link. Pharmacol. Res. 2020, 155, 104722. [Google Scholar] [CrossRef]
  12. Itoh, K.; Ishima, T.; Kehler, J.; Hashimoto, K. Potentiation of NGF-induced neurite outgrowth in PC12 cells by papaverine: Role played by PLC-γ, IP3 receptors. Brain Res. 2011, 1377, 32–40. [Google Scholar] [CrossRef]
  13. Guan, S.; Liu, Q.; Gu, H.; Zhang, Y.Y.; Wei, P.L.; Qi, Y.F.; Liu, J.; Wang, Z. Pluripotent anti-inflammatory immunomodulatory effects of papaverine against cerebral ischemic-reperfusion injury. J. Pharmacol. Sci. 2020, 144, 69–75. [Google Scholar] [CrossRef] [PubMed]
  14. Benej, M.; Hong, X.; Vibhute, S.; Scott, S.; Wu, J.; Graves, E.; Le, Q.T.; Koong, A.C.; Giaccia, A.J.; Yu, B.; et al. Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism. Proc. Natl. Acad. Sci. USA 2018, 115, 10756–10761. [Google Scholar] [CrossRef]
  15. Gao, X.; Yang, Y.; Wang, J.; Zhang, L.; Sun, C.; Wang, Y.; Zhang, J.; Dong, H.; Zhang, H.; Gao, C.; et al. Inhibition of mitochondria NADH-Ubiquinone oxidoreductase (complex I) sensitizes the radioresistant glioma U87MG cells to radiation. Biomed. Pharmacother. 2020, 129, 110460. [Google Scholar] [CrossRef] [PubMed]
  16. Khalifa, S.A.M.; Yosri, N.; El-Mallah, M.F.; Ghonaim, R.; Guo, Z.; Musharraf, S.G.; Du, M.; Khatib, A.; Xiao, J.; Saeed, A.; et al. Screening for natural and derived bio-active compounds in preclinical and clinical studies: One of the frontlines of fighting the coronaviruses pandemic. Phytomedicine 2021, 85, 153311. [Google Scholar] [CrossRef]
  17. Milusheva, M.; Gledacheva, V.; Stefanova, I.; Pencheva, M.; Mihaylova, R.; Tumbarski, Y.; Nedialkov, P.; Cherneva, E.; Todorova, M.; Nikolova, S. In Silico, In Vitro, and Ex Vivo Biological Activity of Some Novel Mebeverine Precursors. Biomedicines 2023, 11, 605. [Google Scholar] [CrossRef]
  18. Heghes, S.C.; Vostinaru, S.C.; Rus, O.; Mogosan, L.M.; Iuga, C. Antispasmodic effect of essential oils and their constituents: A review. Molecules 2019, 24, 1675. [Google Scholar] [CrossRef]
  19. Isyaku, Y.; Uzairu, A.; Uba, S. Computational studies of a series of 2-substituted phenyl-2-oxo-, 2-hydroxyl- and 2-acylloxyethylsulfonamides as potent anti-fungal agents. Heliyon 2020, 6, e03724. [Google Scholar] [CrossRef] [PubMed]
  20. Banerjee, P.; Eckert, A.O.; Schrey, A.K.; Preissner, R. ProTox-II: A webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018, 46, W257–W263. [Google Scholar] [CrossRef]
  21. Mazumder, K.; Hossain, E.; Aktar, A.; Mohiuddin, M.; Sarkar, K.K.; Biswas, B.; Aziz, A.; Abid, A.; Fukase, K. In Silico Analysis and Experimental Evaluation of Ester Prodrugs of Ketoprofen for Oral Delivery: With a View to Reduce Toxicity. Processes 2021, 9, 2221. [Google Scholar] [CrossRef]
  22. Anzali, S.; Barnickel, G.; Cezanne, B.; Krug, M.; Filimonov, D.; Poroikov, V. Discriminating between Drugs and Nondrugs by Prediction of Activity Spectra for Substances (PASS). J. Med. Chem. 2001, 44, 2432–2437. [Google Scholar] [CrossRef] [PubMed]
  23. Mathew, B.; Suresh, J.; Anbazhagan, S. Synthesis and PASS-assisted in silico approach of some novel 2-substituted benzimidazole bearing a pyrimidine-2,4,6(trione) system as mucomembranous protector. J. Pharm. Bioallied Sci. 2013, 5, 39–43. [Google Scholar] [CrossRef] [PubMed]
  24. Ekins, S.; Olechno, J.; Williams, A.J. Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses. PLoS ONE 2013, 8, e62325. [Google Scholar] [CrossRef] [PubMed]
  25. Milusheva, M.; Gledacheva, V.; Stefanova, I.; Feizi-Dehnayebi, M.; Mihaylova, R.; Nedialkov, P.; Cherneva, E.; Tumbarski, Y.; Tsoneva, S.; Todorova, M.; et al. Synthesis, Molecular Docking, and Biological Evaluation of Novel Anthranilic Acid Hybrid and Its Diamides as Antispasmodics. Int. J. Mol. Sci. 2023, 24, 13855. [Google Scholar] [CrossRef] [PubMed]
  26. Docheva, M.; Dagnon, S.; Statkova-Abeghe, S. Flavonoid content and radical scavenging potential of extracts prepared from tobacco cultivars and waste. Nat. Prod. Res. 2014, 28, 1328–1334. [Google Scholar] [CrossRef] [PubMed]
  27. Talley, N.J. Drug therapy options for patients with irritable bowel syndrome. Am. J. Manag. Care 2001, 7, S261–S267. [Google Scholar] [PubMed]
  28. Milusheva, M.; Gledacheva, V.; Batmazyan, M.; Nikolova, S.; Stefanova, I.; Dimitrova, D.; Saracheva, K.; Tomov, D.; Chaova-Gizdakova, V. Ex Vivo and In Vivo Study of Some Isoquinoline Precursors. Sci. Pharm. 2022, 90, 37. [Google Scholar] [CrossRef]
  29. Daina, A.; Michielin, O.; Zoete, V. Swiss ADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [Google Scholar] [CrossRef]
  30. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2001, 46, 3–26. [Google Scholar] [CrossRef]
  31. Martin, Y.C. A bioavailability score. J. Med. Chem. 2005, 48, 3164–3170. [Google Scholar] [CrossRef]
  32. Kevin, B.; Robert, W.; Iain, G.; Kevin, D. Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: Challenges to the discovery scientist. Curr. Drug Metab. 2003, 4, 461–485. [Google Scholar] [CrossRef]
  33. Mishra, S.; Dahima, R. In vitro ADME studies of TUG-891, a GPR-120 inhibitor using Swiss ADME predictor. J. Drug Deliv. Ther. 2019, 9, 366–369. [Google Scholar] [CrossRef]
  34. Itoh, T. Pharmacomechanical Coupling in Vascular Smooth Muscle Cells—An Overview. Jpn. J. Pharmacol. 1991, 55, 1–9. [Google Scholar] [CrossRef] [PubMed]
  35. Somlyo, A.V.; Somlyo, A.P. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmacol. Exp. Ther. 1968, 159, 129–145. [Google Scholar] [PubMed]
  36. Ito, M.; Nakano, T.; Erdodi, F.; Hartshorne, D.J. Myosin phosphatase: Structure, regulation and function. Mol. Cell. Biochem. 2004, 259, 197–209. [Google Scholar] [CrossRef] [PubMed]
  37. Droogmans, G.; Casteels, R. Electromechanical and Pharmacomechanical Coupling in Vascular Smooth Muscle. In Physiology and Pathophysiology of the Heart Developments in Cardiovascular Medicine; Sperelakis, N., Ed.; Springer: Boston, MA, USA, 1989; Volume 90. [Google Scholar] [CrossRef]
  38. Daniluk, J.; Malecka-Wojciesko, E.; Skrzydlo-Radomanska, B.; Rydzewska, G. The Efficacy of Mebeverine in the Treatment of Irritable Bowel Syndrome—A Systematic Review. J. Clin. Med. 2022, 11, 1044. [Google Scholar] [CrossRef] [PubMed]
  39. Szymaszkiewicz, A.; Zieli’nska, M. Irritable bowel syndrome: Current therapies and future perspectives. In A ComprehensiveOverview of Irritable Bowel Syndrome; Fichna, J., Ed.; Elsevier: London, UK, 2020; pp. 129–144. ISBN 978-0-12-821324-7. [Google Scholar] [CrossRef]
  40. Mustafa, S.M.; Thulesius, O. Cooling-induced gastrointestinal smooth muscle contractions in the rat. Fundam. Clin. Pharmacol. 2001, 15, 349–354. [Google Scholar] [CrossRef]
  41. Bitar, K.N. Function of gastrointestinal smooth muscle: From signaling to contractile proteins. Am. J. Med. 2003, 115 (Suppl. 3A), 15–23. [Google Scholar] [CrossRef]
Biomedicines 12 01556 g001
Figure 1. Structures of mebeverine (a), papaverine (b), and target 3-isopropyl-3,4-dihydroisoquinolines (c).
Figure 1. Structures of mebeverine (a), papaverine (b), and target 3-isopropyl-3,4-dihydroisoquinolines (c).
Biomedicines 12 01556 g001
Biomedicines 12 01556 sch001
Scheme 1. Cyclization of amides 4 to 1,3-disubstituted-3,4-dihydroisoquinolines 5.
Scheme 1. Cyclization of amides 4 to 1,3-disubstituted-3,4-dihydroisoquinolines 5.
Biomedicines 12 01556 sch001
Biomedicines 12 01556 g002
Figure 2. A graphical depiction outlining the procedure for examining the mechanical functions of isolated smooth muscle preparations. This includes illustrative traces for compound 5d compared to 5b and was generated using BioRender.com (accessed on 29 January 2024).
Figure 2. A graphical depiction outlining the procedure for examining the mechanical functions of isolated smooth muscle preparations. This includes illustrative traces for compound 5d compared to 5b and was generated using BioRender.com (accessed on 29 January 2024).
Biomedicines 12 01556 g002
Biomedicines 12 01556 g003aBiomedicines 12 01556 g003b
Figure 3. Changes in the smooth muscle tissue’s (A) tonus, (B) amplitude, and (C) frequency induced by compounds 4a–d compared to those of 5a–d (5 × 10−5 mol/L) (n = 12). Statistically significant differences: * p < 0.05, comparison is between the measured value and mebeverine as a control; # p < 0.05, comparison is between the measured values of precursors to isoquinolines, 4a vs. 5a, 4b vs. 5b, 4c vs. 5c, and 4d vs. 5d.
Figure 3. Changes in the smooth muscle tissue’s (A) tonus, (B) amplitude, and (C) frequency induced by compounds 4a–d compared to those of 5a–d (5 × 10−5 mol/L) (n = 12). Statistically significant differences: * p < 0.05, comparison is between the measured value and mebeverine as a control; # p < 0.05, comparison is between the measured values of precursors to isoquinolines, 4a vs. 5a, 4b vs. 5b, 4c vs. 5c, and 4d vs. 5d.
Biomedicines 12 01556 g003aBiomedicines 12 01556 g003b
Biomedicines 12 01556 g004
Figure 4. Antioxidant activity of 5d compared to those of mebeverine, rutin, and quercetin as positive controls.
Figure 4. Antioxidant activity of 5d compared to those of mebeverine, rutin, and quercetin as positive controls.
Biomedicines 12 01556 g004
Table 1. ADME parameters of compounds 5a–d representing drug-likeness.
Table 1. ADME parameters of compounds 5a–d representing drug-likeness.
CompoundMW, g/molXLOGP3ESOL logSFraction Csp3RBBA ScoreSA ScoreLD50, mg/kg
5a187.283.01−3.150.4610.553.12240
5b249.354.66−4.660.2820.553.49240
5c283.85.29−5.240.2820.553.53240
5d263.384.60−4.620.3230.553.63240
MW: molecular weight, XLOGP3: octanol/water partition coefficient, ESOL logS: estimated aqueous solubility, fraction Csp3: ratio of sp3-hybridized carbons over the total carbon count, RB: rotatable bonds, BA: bioavailability, SA: synthetic accessibility, predicted LD50: lethal dose to 50% of animals.
Table 2. Changes in the parameters of SCA of gastric SMPs under the influence of 4a–d compared to 5a–d. All data are expressed as mean values ± standard error of the mean.
Table 2. Changes in the parameters of SCA of gastric SMPs under the influence of 4a–d compared to 5a–d. All data are expressed as mean values ± standard error of the mean.
SCA Parameters Control5a4a5b4b5c4c5d4d
tonus, mN0.91 ± 0.16−0.96 ± 0.11−0.50 ± 0.06−1.04 ± 0.190−0.91 ± 0.040.88 ± 0.083.53 ± 0.11−2.03 ± 0.13
frequency, min−15.07 ± 0.097.12 ± 0.054.98 ± 0.057.33 ± 0.0306.88 ± 0.135.01 ± 0.245.78 ± 0.074.79 ± 0.08
amplitude, mN2.85 ± 0.222.77 ± 0.172.68 ± 0.184.18 ± 0.0802.55 ± 0.162.50 ± 0.096.66 ± 0.041.45 ± 0.20
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Milusheva, M.; Stoyanova, M.; Gledacheva, V.; Stefanova, I.; Todorova, M.; Nikolova, S. Spasmolytic Activity of 1,3-Disubstituted 3,4-Dihydroisoquinolines. Biomedicines 2024, 12, 1556. https://doi.org/10.3390/biomedicines12071556

AMA Style

Milusheva M, Stoyanova M, Gledacheva V, Stefanova I, Todorova M, Nikolova S. Spasmolytic Activity of 1,3-Disubstituted 3,4-Dihydroisoquinolines. Biomedicines. 2024; 12(7):1556. https://doi.org/10.3390/biomedicines12071556

Chicago/Turabian Style

Milusheva, Miglena, Mihaela Stoyanova, Vera Gledacheva, Iliyana Stefanova, Mina Todorova, and Stoyanka Nikolova. 2024. "Spasmolytic Activity of 1,3-Disubstituted 3,4-Dihydroisoquinolines" Biomedicines 12, no. 7: 1556. https://doi.org/10.3390/biomedicines12071556

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop