Synthesis and Biological Evaluation of Tetrahydroisoquinoline Derivatives as Trypanocidal Agents

Abstract

American trypanosomiasis is a parasitic illness of major public health relevance, resulting from infection with the protozoan Trypanosoma cruzi and predominantly impacting populations in low-resource settings. Current treatments, benznidazole and nifurtimox, are limited by their efficacy in the chronic phase, toxicity, and side effects, necessitating the search for new therapeutic agents. Cruzain, a key protease for parasite survival and infection, is a validated drug target. This work involved the synthesis and characterization of novel amides derived from 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Their activity was evaluated against both cruzain and T. cruzi. The hydrochloride salts 4a and 4b showed moderate cruzain inhibition (60.2 ± 2.4% and 69.3 ± 2.6% inhibition at 100 μM, respectively). Notably, compound 3d and its hydrochloride salt 4d demonstrated significant antiparasitic activity with IC₅₀ values of 10.5 and 13.7 μM, respectively. However, their low cruzain inhibition (∼15%) suggests that their mechanism of action is likely through a different biological target.

1. Introduction

Infection with the protozoan Trypanosoma cruzi gives rise to a persistent disorder that affects multiple organs and represents a major challenge in parasitic disease research. Transmission to humans occurs mainly through exposure to the feces of infected insects belonging to the Triatominae subfamily, popularly known as kissing bugs. The disease initially presents with an acute phase that lasts for approximately two months following infection and is marked by high levels of parasitemia, often accompanied by absent or nonspecific clinical symptoms, and a late chronic phase, in which the protozoan is found in its intracellular form, primarily in the muscle cells of the heart and gastrointestinal system. After a period of 10–30 years, about a third of infected individuals develop symptoms related to cardiac disorders and about a tenth develop digestive, neurological, or mixed changes. These changes are potentially fatal, primarily due to the destruction of the nervous system and heart muscle, resulting in arrhythmia and progressive heart failure.

The World Health Organization has recognized Chagas disease since 2005 as 1 of the 20 conditions classified as neglected tropical diseases. It is estimated that approximately 6–7 million individuals worldwide are infected with T. cruzi, with the main affected regions being endemic areas in 21 Latin American countries. , According to the drugs for neglected diseases initiative (DNDi), approximately 100 million people are at risk of infection, and an estimated 40,000 new cases occur annually, of which only 10% are diagnosed. Furthermore, Chagas disease is responsible for 10,000 deaths annually, making it the parasitic disease that causes the most deaths annually in Latin America.

Currently, treatment of T. cruzi infection relies exclusively on the nitroheterocyclic drugs benznidazole (BZN) and nifurtimox (NFX), which are recommended for use in cases of acute infections (including newborns or infants infected through congenital transmission), reactivation during the chronic phase, chronic patients up to 18 years of age, and infected women of reproductive age. In addition to their use being limited to acute infections, other limitations of these drugs include prolonged treatment (60–90 days), contraindication during pregnancy due to genotoxic effects, and the occurrence of adverse events that result in significant treatment discontinuation rates. Therefore, the search for new treatment strategies/regimens is justified, as well as the search for new drugs capable of treating patients in both phases of the disease, in a shorter time frame, and providing a better safety profile.

Among the parasite’s molecular targets considered for the development of new anti-T. cruzi agents, the protease cruzain (also known as Cruzipain) is particularly noteworthy. It is a cysteine protease initially expressed as a zymogen in all stages of the parasite’s development and in this context plays a central role in multiple biological functions, including metabolic support, regulation of immune cell activity, and facilitation of entry into host tissues.

Via a docking-based virtual screening, Santos et al. identified the commercially acquired racemic mixture of furfurylamide of R and S-1,2,3,4-tetrahydroisoquinolin-3-carboxylic acids (THIQA) as cruzain-competitive inhibitors. Thus, the present work aims to synthesize new derivatives of this acid and to evaluate their potential to inhibit cruzain as well as their trypanocidal activity.

2. Results and Discussion

To synthesize the intended THIQA amides, the first synthetic step consisted of protection of the amine group of the tetrahydroisoquinoline ring, in order to avoid cross side reactions during the respective amides’ formation. For this purpose, (Boc)₂O was employed in alkaline medium, with THF as the solvent (Scheme ).

1. Synthesis Route Used for Preparation of THIQA Amides .

^a Reaction conditions: (i) NaOH_(aq) 1 mol/L, (Boc)₂O, THF; (ii) DMAP, EDAC, respective amine, DCM; (iii) TFA, 0 °C, DCM; (iv) HCl_(aq) 1 mol/L, acetone, 0 °C.

¹H and ¹³C nuclear magnetic resonance (NMR) data confirmed the presence of the Boc group, indicating the successful protection of the amine nitrogen atoms in both THIQAs, affording 1a (S-enantiomer) and 1b (R-enantiomer) in excellent yields. It is worth highlighting that the characteristic signals for tert-butyl N-Boc groups are found duplicate in their respective NMR spectra. This phenomenon may be explained by the conformation variations of the N-Boc groups, and for that, in silico conformational analyses were performed.

The initial conformational analysis of 1a and 1b generated 8 conformers and 10 low-energy conformers using the molecular mechanics level of theory, respectively. After optimizing the geometry of all conformers with PM6 and density functional theory (DFT) levels of theory, we ordered the obtained conformations according to the calculated energies from the lowest to highest. At this point, we analyzed the differences between conformers and found two major conformational differences (molecular motions) for both compounds: (i) a “breathing” movement of the aliphatic ring (Figure , vertical transitions) similar to a boat-chair transition and (ii) the rotation of the nitrogen and carbonylic carbon atom bond (Figure , horizontal transitions). The other conformers (not shown) are just minor variations of the carboxylic acid position and intermediates of the “breathing” transition (one carbon positioned in front of the ring and the nitrogen atom behind, or vice versa, present only in 1b). Interestingly, the first movement ranges between 0.33 and 0.62 kcal/mol means that they practically coexist in energy terms. The second movement occurs in an energy window lower than 5 kcal/mol. Therefore, both movements contribute to the tert-butyl position and affect the NMR signals.

1.

Conformational analysis of 1a and 1b (A and B, respectively) showing the most representative conformers and their transition energies.

The second step of the proposed synthetic route consisted of the formation of amides 2a–f with different amines, using EDAC as a coupling reagent and DMAP as a catalyst, with yielding ranging from 72 to 96%, as shown in Scheme .

The presence of the amide side chains also contributes to the duplication of N-Boc tert-butyl signals. Exemplified with 4-methylpiperazine amide 2e, this compound generated 34 conformers at the molecular mechanics level of theory, as expected due to the larger and more flexible structure. We analyzed the 10 first lowest energy conformers also calculated with PM6 followed by DFT (Figure ), which ranged 1.33 kcal/mol, suggesting the easy conversion between them. The main observed molecular movements are related to the flip of the amide bond of the piperazine ring. This motion indirectly influenced the position of the tert-butyl group. Furthermore, the minor conformation within this sampling comprised the flipping of the carbamate N-carbonyl bond with a lower-energy transition than that of 1a and 1b (1.24 kcal/mol). Both conformational effects change the position of the tert-butyl group and could affect the NMR spectra, generating the observed duplicated signals.

2.

Conformational analysis of 2e: superimposition of the 10 lowest energy conformers (A, major conformation is represented in dark gray carbons, and the minor conformation is represented in light gray carbons), the seven conformers with the conserved N-carbonyl bond (B) and the three others with this bond flipped (C). The transition between two states of the carbamate N-carbonyl bond and their transition energy (D).

Besides the duplicated specious NMR spectra due to the conformer equilibrium of N-Boc groups, another noteworthy feature related to the characterization of the synthesized compounds is the absence of a protector group in the mass spectra. With the exception of 2e and 2f, all the N-Boc compounds had lost their carbamoyl moiety in-source during the ESI-QTOF mass spectrometer analysis, leading to detection of the corresponding free tetrahydroisoquinoline amines only. Although electrospray ionization is well known to be a milder technique when it comes to fragmentation in comparison to harder ionization, for example, electron impact, this phenomenon may occur, especially to labile N-carbonyl bonds, such as the synthesized substances in the present work.

As the third step of the adopted synthetic route to obtain unprotected tetrahydroisoquinoline rings, the synthesized amides 2a–f were subjected to reaction with trifluoroacetic acid (TFA) at low temperature in dichloromethane, affording 3a–f as the final amides. Additionally, the hydrochlorides of furfuryl, benzyl, and 3-phenylpropylamides were obtained (4a–d) (Figure ). This strategy is based on the improvement of their hydrosolubility and potential gain of new ionic molecular interactions (concerning the N-heteroatom).

3.

Chemical structures of synthesized hydrochloride salts.

Substances 3a–f were assessed for their inhibitory potential toward cruzain and for their effects on intracellular amastigotes and bloodstream trypomastigotes of the β-galactosidase-expressing Tulahuen strain of T. cruzi. Compounds were initially evaluated in the cruzain assay after 10 min of preincubation with the enzyme (Table ). The hydrochloride salts 4a and 4b exhibited the highest inhibition percentages, whereas all other analogues were essentially inactive. The compounds were subsequently tested without preincubation, and the inhibition values under both conditions were compared to assess time-dependent inhibition, typically associated with slow-binding or covalent inhibitors. However, no meaningful time-dependent inhibition was observed for any compounds, suggesting noncovalent binding.

1. Substance Inhibition against Cruzain.

substance	Cruzain inhibition at 100 μM (mean ± SEM %)
	0′	10′ inc
3a	9.3 ± 1.7	16.3 ± 0.8
3b	ND	6.4 ± 0.7
3c	ND	14.1 ± 1.9
3d	ND	14.1 ± 3.5
3e	ND	8.4 ± 2.8
3f	ND	9.6 ± 4.0
4a	55.0 ± 1.0	60.2 ± 2.4
4b	47.9 ± 5.3	69.3 ± 2.6
4c	ND	14.5 ± 3.8
4d	ND	15.0 ± 2.8
E64	73.4 ± 3.1	100 ± 5.3

^aND – nondetermined.

^bInhibition of cruzain at 100 μM for each compound following a 10 min preincubation with the enzyme (10′) or without preincubation (0′). Mean over two independent experiments, in triplicate ± standard error of the mean.

^cHydrochloride salt.

^dE64 – epoxy succinate (positive control of inhibition).

Analysis of the structure–activity relationship (SAR) demonstrated that the tetrahydroisoquinoline and furan rings are important for enzyme inhibition, which likely accounts for the low activity of compounds 3c–f, 4c, and 4d. Although 4a and 4b were active, the free bases (3a and 3b) showed very low inhibition, suggesting that the positively charged amino group may be important for activity or solubility under the assay conditions.

In previous work, the commercially acquired racemic mixture of THIQA-furfurylamides (4a + 4b) was reported as a potent competitive cruzain inhibitor (100 ± 2% inhibition at 100 μM; IC₅₀ = 3.0 ± 2.0 μM). Surprisingly, in the present study, the pure synthesized and characterized isomers exhibited lower potencies, with IC₅₀ values of 90 ± 13 μM for 4a and 53 ± 5 μM for 4b (Figure ). Due to this result, we also tested a 1:1 mixture of the two isomers; however, we observed intermediate cruzain inhibition values when compared to individual isomers. Thus, the difference observed cannot be explained based on any synergistic effect involving the isomers and might be related to unknown differences between the commercially acquired samples and the ones synthesized in this study.

4.

IC₅₀ curves for compounds 4a and 4b against cruzain. Curves were obtained with a 10 min (10′) preincubation of the compound with the enzyme. Two independent experiments were performed: experiment 1 (blue curves) and experiment 2 (black curves).

In the phenotypic assays against T. cruzi, L929 fibroblasts were infected with 10 trypomastigotes per cell, and the intracellular infection was allowed to progress for 2 days. During this period, internalized trypomastigotes differentiated into intracellular amastigotes, ensuring that both early intracellular trypomastigotes and replicative amastigotes were present in the culture. The test compounds were then added for an additional four day incubation period, allowing the determination of inhibitory activity across the full intracellular developmental cycle of the parasite. Thus, the IC₅₀ values reported here represent the compounds’ cumulative phenotypic activity against both intracellular trypomastigotes and amastigotes. The compounds were tested in serial dilutions prepared from freshly made stock solutions (20 mg/mL in DMSO). The results of the anti-T. cruzi assays are summarized in Table . IC₅₀ and CC₅₀ values were calculated only for compounds that exhibited ≥ 70% inhibition at 100 μg/mL, following the standard screening cascade for T. cruzi drug discovery.

2. Substance Percentage of Inhibition of Parasitic Growth and Cell Death.

substance	IC50 (μM)	CC50 (μM)	SI
3a	ND	ND	ND
3b	ND	ND	ND
3c	211.4 ± 23.6	478.0	2.2
3d	10.5 ± 0.3	184.1	17.5
3e	ND	ND	ND
3f	ND	ND	ND
4a	79.6 ± 0.0	ND	ND
4b	32.4 ± 0.0	ND	ND
4c	227.5 ± 12.2	210.0	0.9
4d	13.7 ± 0.0	390.8	28.5
benznidazole	3.8	2401.7	625

^aIC₅₀, concentration of substance that reduces parasitic growth by 50%.

^bCC₅₀, concentration of substance that induces 50% cell death (L929).

^cSI, selectivity index, calculated by the ratio CC₅₀/ IC₅₀.

^dND, nondetermined.

^eHydrochloride salt.

The most promising results were obtained for substances 3d and 4d with IC₅₀ values of 10.5 ± 0.3 and 13.7 ± 0.0 μM, respectively, as well as for 4b, which exhibited an IC₅₀ of 32.4 ± 0.0 μM. Additionally, substances 3d and 4d presented selectivity indices significantly higher than those of the other substances, with values of 17.5 and 28.5, respectively. In contrast, substances 3d and 4d presented low inhibitory activity against cruzain (14.1 ± 3.5% for the free base and 15.0 ± 2.8% for the hydrochloride), suggesting that their antiparasitic effects may be mediated through alternative mechanisms. Substances 3c and 4c exhibited low activity, with IC₅₀ values of 211.4 ± 23.6 and 227.5 ± 12.2, respectively. They also displayed extremely low selectivity indices (0.9 to 2.2), indicating that although they showed some inhibitory effect on T. cruzi growth, they also induced marked cytotoxicity, suggesting an unfavorable toxicity profile.

Although cruzain was initially evaluated as a potential molecular target, the absence of inhibitory activity for the most active compounds suggests that this protease is unlikely to be the primary target responsible for the observed anti-T. cruzi effects. At this stage, the molecular target underlying the intracellular activity remains to be elucidated, and proposing a specific mechanism of action would be speculative without further experimental validation. Alternative targets relevant to Chagas disease drug discovery include proteases other than cruzain, the sterol biosynthesis pathway, the antioxidant defense system, the mitochondrial electron transport chain, and pathways involved in mRNA processing, as discussed in a recent review by our group.

3. Experimental Section

3.1. Chemistry

The NMR analyses were carried out by using a Bruker AVANCE NEO spectrometer operating at 600 MHz (Bruker, MA, USA). Tetramethylsilane served as the internal standard. Chemical shifts are reported as δ values in parts per million (ppm), while spin–spin coupling constants (J-values) are expressed in hertz (Hz). Signal multiplicities were described as s (singlet), bs (broad singlet), d (doublet), dd (double doublet), t (triplet), td (triplet of doublets), qt (quintet), or m (multiplet). Compounds 1a, 1b, 2c and 3c, and 2e and 3e have been previously reported in the literature, and their synthetic routes were reproduced with minor adaptations based on the described procedures.

UHPLC–HRMS/MS experiments were carried out using a Nexera UPLC-system (Shimadzu) coupled to a maXis ESI-QTOF high-resolution mass spectrometer (Bruker), controlled with Compass 1.7 software (Bruker). Chromatographic separation was achieved on a Shim-Pack XR-ODS-III reversed-phase column (C18, 2.2 μm, 2.0 × 150 mm) at 40 °C, with a constant flow of 0.40 mL min^–1. Elution was performed with a binary solvent system composed of Milli-Q water containing 0.1% formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B), using the following gradient: 5% B maintained for 0.5 min, a linear increase to 100% B over 10 min, and an isocratic step at 100% B for an additional 1.5 min. UV-PDA detection was conducted in the 190–450 nm range, after which mass spectra were recorded in positive ion mode over an m/z interval of 100–1500, with a spectral acquisition frequency of 5 Hz. The electrospray source was operated with an end-plate offset of 500 and 4500 V capillary voltage. Nebulization was achieved at 3.0 bar, while dry gas flow was delivered at 8 L min^–1 and maintained at 200 °C. Fragmentation data were recorded in a data-dependent acquisition mode using collision energies ranging from 15 to 60 eV. Mass accuracy was ensured through external calibration performed by direct infusion of 20 μL of a 1 mM sodium formate solution prepared in 50% 2-propanol, followed by postacquisition recalibration of the acquired data sets. Compound detection was performed using chromatographic peak dissection with subsequent formula determination based on exact mass and isotope pattern.

All reagents were obtained from Sigma-Aldrich (Missouri, USA) and employed as received, without additional purification steps.

3.1.1. General Methodology for the Synthesis of Substances 2a–2f

To a 50 mL round-bottom flask was added 1 equiv of the N-Boc-THIQA (1a or 1b), 1 equiv of respective amine, and catalytic amount of 4-dimethylaminopyridin (DMAP) and dichloromethane. The mixture was kept under magnetic stirring and 0 °C and then 1 equiv of 1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide hydrochloride (EDAC.HCl). The reaction was kept under low temperature for 20 min; then, ice bath was discontinued, and the mixture was allowed to warm to ambient conditions. Reaction progress was followed by thin-layer chromatography using an ethyl acetate/hexane (1:1, v/v) solvent system, with visualization performed by using iodine vapor and CAM staining. Once the starting material was no longer detected, dichloromethane (15 mL) was added, and the reaction mixture was transferred to a separatory funnel. The organic phase was subsequently washed with distilled water in three successive portions of 15 mL. The organic layer was dried over anhydrous Na₂SO₄ filtered, and the solvent was removed under reduced pressure, obtaining the desired product.

3.1.2. tert-Butyl (S)-3-((Furan-2-ylmethyl)­carbamoyl)­3,4-dihydroisoquinoline-2­(1H)-carboxylate (2a)

According to the general methodology described in 3.1.1, the reaction was conducted using 505 mg (1.8 mmol) of carboxylic acid 1a, 177 μL (194 mg, 2 mmol, 1.1 equiv) of furfurylamine, 3 mg of DMAP, and 5 mL of dichloromethane. After the reaction mixture reached 0 °C, 286 mg (1.5 mmol, 0.82 equiv) of EDAC.HCl was added. The reaction was kept under an inert N₂ atmosphere, magnetic stirring at room temperature for 5 h, to obtain 467 mg (72% yield) of 2a as an orange pasty solid. [α]_D (MeOH, 1% w/v): −22°; ¹H NMR (600 MHz, CDCl₃), δ/ppm: 7.18–7.03 (m, 5H, H-1, H-2, H-3, H-6 and H-15); 6.15 (m, 1H, H-14); 5.86 (s, 1H, H-13); 4.80–4.12 (m, 5H, H-7, H-8 and H-11); 3.24–3.00 (m, 2H, H-9); 2.25–2,17 (m, 1H, CONH); 1.42–1.32 (m, 9H, H-18, H-19 and H-20); ¹³C NMR (150 MHz, CDCl₃), δ/ppm: 142.2 (C-15); 134.0 (C-4 or C-5); 128.0–126.3 (C-1, C-2, C-3 and C-6); 110.5 (C-13 and C-14); 81.5 (C-17); 56.9 (C-8); 44.7 (C-7); 36.3 (C-11); 32.1 (C-9); 28.6 and 28.4 (C-18, C-19 and C-20); HRMS (m/z) [M + H]⁺ calcd for C₁₅H₁₇N₂O₂ ⁺, 257.1285 (without Boc group); found, 257.1287.

3.1.3. tert-Butyl (R)-3-((Furan-2-ylmethyl)­carbamoyl)­3,4-dihydroisoquinoline-2­(1H)-carboxylate (2b)

According to the general methodology described in 3.1.1, the reaction was conducted using 496 mg (1.8 mmol) of carboxylic acid 1b, 160 μL (176 mg, 1.8 mmol, 1 equiv) of furfurylamine, 10 mg of DMAP, and 7 mL of dichloromethane. After the reaction mixture reached 0 °C, 279 mg (1.5 mmol, 0.8 equiv) of EDAC.HCl was added. The reaction was kept under magnetic stirring at room temperature for 5 h to obtain 510 mg (80% yield) of 2b as an orange pasty solid. [α]_D (MeOH, 1% w/v): +20°; ¹H NMR (600 MHz, CDCl₃), δ/ppm: 7.25–7.10 (m, 5H, H-1, H-2, H-3, H-6 and H-15); 6.23 (m, 1H, H-14); 5.93 (s, 1H, H-13); 4.88–4.20 (m, 5H, H-7, H-8 and H-11); 3.33–3.03 (m, 2H, H-9); 2.26–2.16 (m, 1H, CONH); 1.50–1.40 (m, 9H, H-18, H-19 and H-20); ¹³C NMR (150 MHz, CDCl₃), δ/ppm: 142.2 (C-15); 134.0 (C-4 or C-5); 128.0–126.4 (C-1, C-2, C-3 and C-6); 110.4 (C-13 and C-14); 81.5 (C-17); 56.9 (C-8); 44.7 (C-7): 36.4 (C-11); 32.2 (C-9); 28.6 and 28.4 (C-18, C-19 and C-20); HRMS (m/z) [M + H]⁺ calcd for C₁₅H₁₇N₂O₂ ⁺, 257.1285 (without Boc group); found, 257.1284.

3.1.4. tert-Butyl (S)-3-(Benzylcarbamoyl)-3,4-dihydroisoquinoline-2­(1H)-carboxilate (2c)

According to the general methodology described in 3.1.1, the reaction was conducted using 752 mg (2.7 mmol) of carboxylic acid 1a, 300 μL (294 mg, 2.7 mmol, 1 equiv) of benzylamine, 5 mg of DMAP, and 7 mL of dichloromethane. After the reaction mixture reached 0 °C, 425 mg (2.2 mmol, 0.8 equiv) of EDAC.HCl was added. The reaction was kept under magnetic stirring at room temperature for 6 h to obtain 740 mg (74% yield) of 2c as a yellow pasty solid. [α]_D (MeOH, 1% w/v): −20°; ¹H NMR (600 MHz, CDCl₃), δ/ppm: 7.19–6.72 (m, 9H, H-1, H-2, H-3, H-6, H-13, H-14, H-15, H-16 and H-17); 4.84–4.11 (m, 5H, H-7, H-8 and H-11); 3.31–2.94 (m, 2H, H-9); 2.18–2.06 (m, 1H, CONH): 1.42–1.35 (m, 9H, H-20, H-21, and H-22); ¹³C NMR (150 MHz, CDCl₃), δ/ppm: 134.0 (C-4 or C-5); 128.6–126.4 (C-1, C-2, C-3, C-6, C-13, C-14, C-15, C-16, and C-17); 81.4 (C-19); 56.9 (C-8); 43.2 (C-7 or C-11); 32.3 (C-9); 28.5 and 28.4 (C-20, C-21 and C-22); HRMS (m/z) [M + H]⁺ calc for C₁₇H₁₉N₂O⁺, 267.1492 (without Boc group); found, 267.1497.

3.1.5. tert-Butyl (S)-3-((3-Phenylpropyl)­carbamoyl)-3,4-dihydroisoquinoline-2­(1H)-carboxilate (2d)

According to the general methodology described in 3.1.1, the reaction was conducted using 505 mg (2.7 mmol) of the carboxylic acid 1a, 260 μL (247 mg, 1.8 mmol, 1 equiv) of 3-phenyl-1-propylamine, 6 mg of DMAP, and 8 mL of dichloromethane. After the reaction mixture reached 0 °C, 353 mg (1.8 mmol, 1 equiv) of EDAC.HCl was added. The reaction was kept under magnetic stirring at room temperature for 5 h to obtain 691 mg (96% yield) of 2d as a yellow pasty solid. [α]_D (MeOH, 1% w/v): −12°; ¹H NMR (600 MHz, acetone-d ₆), δ/ppm: 7.14–6.96 (m, 9H, H-1, H-2, H-3, H-6, H-15, H-16, H-17, H-18, H-19); 4.98–4.34 (m, 2H, H-7 and H-8); 3.26–2.81 (m, 7H, H-7, H-9, H-11, and H-13); 2.36–2.29 (m, 2H, H-12); 1.96 (s, 1H, CONH); 1.41–1.29 (m, 9H, H-22, H-23, and H-24); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 143.3 (C-14); 133.9–126.8 (C-1, C-2, C-3, C-6, C-15, C-16, C-17, C-18, and C-19); 81.8 and 80.8 (C-21); 58.7 and 57.3 (C-8); 46.2 and 45.4 (C-7); 39.9 and 39.7 (C-11); 33.8–30.7 (C-12, C-13, and C-9); 28.9–28.6 (C-22, C-23, and C-24); HRMS (m/z) [M + H]⁺ calcd for C₁₉H₂₃N₂O⁺, 295.1805 (without Boc group); found, 295.1805.

3.1.6. tert-Butyl (S)-3-(4-Methylpiperazine-1-carbonyl)-3,4-dihydroisoquinoline-2­(1H) Carboxylate (2e)

According to the general methodology described in 3.1.1, the reaction was conducted using 755 mg (2.7 mmol) of carboxylic acid 1a, 310 μL (280 mg, 2.8 mmol, 1 equiv) of 1-methylpiperazine, 5 mg of DMAP, and 7 mL of dichloromethane. After the reaction mixture reached 0 °C, 526 mg (2.7 mmol, 1 equiv) of EDAC.HCl was added. The reaction was kept under magnetic stirring at room temperature for 5 h to obtain 927 mg (95% yield) of 2e as a yellow pasty solid. [α]_D (MeOH, 1% w/v): −34°; ¹H NMR (600 MHz, CDCl₃), δ/ppm: 7.24–7.04 (m, 4H, H-1, H-2, H-3, and H-6); 5.27 (m, 0.5H, H-8); 4.90–4.79 (m, 1.5H, H-8 and H-7); 4.43–4.34 (m, 1H, H-7); 3.64–3.46 (m, 4H, H-11, and H-14); 3.01–2.92 (m, 2H, H-9); 2.40–2.07 (m, 7H, H-12, H-13, and H-15); 1.45–1.41 (m, 9H, H-18, H-19, and H-20); ¹³C NMR (150 MHz, CDCl₃), δ/ppm: 133.5 (C-4 or C-5); 128.5–125.9 (C-1, C-2, C-3, and C-6); 80.8 (C-17); 55.4 and 55.0 (C-12 and C-13); 52.3 and 49.9 (C-8); 46.2 (C-15); 45.6, 44.8, 44.3, and 42.2 (C-7, C-11, and C-14); 31.4 and 30.7 (C-9); 28.6 (C-18, C-19, C-20); HRMS (m/z) [M + H]⁺ calcd for C₂₀H₃₀N₃O₃ ⁺, 360.2282; found, 360.2281.

3.1.7. tert-Butyl (S)-3-((3-Morpholinopropyl)­carbamoyl)-3,4-dihydroisoquinoline-2­(1H) Carboxylate (2f)

According to the general methodology described in 3.1.1, the reaction was conducted using 519 mg (1.9 mmol) of carboxylic acid 1a, 274 μL (270 mg, 1.9 mmol, 1 equiv) of morpholino-1-propylamine, 10 mg of DMAP, and 8 mL of dichloromethane. After the reaction mixture reached 0 °C, 359 mg (1.9 mmol, 1 equiv) of EDAC.HCl was added. The reaction was kept under magnetic stirring at room temperature for 21 h to obtain 708 mg (94% yield) of 2f as a yellow pasty solid. [α]_D (MeOH, 1% w/v): −12°; ¹H NMR (600 MHz, acetone-d ₆), δ/ppm: 7.12–7.05 (m, 4H, H-1, H-2, H-3, and H-6); 4.66–4.32 (m, 2H, H-7, and H-8); 3.46–3.43 (m, 3H, H-15 or H-16 and H-7); 3.17–2.94 (m, 6H, H-9, H-11 and H-15 or H-16); 2.16–1.94 (m, 7H, H-13, H-14, H-17, and CONH); 1.92 (qt, J _11,12 and J _12,13 = 2.25 Hz, 2H, H-12); 1.40–1,30 (m, 9H, H-20, H-21, H-22); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 128.9–127.2 (C-1, C-2, C-3, and C-6); 80.8 (C-19); 67.7 (C-15 and C-16); 64.5 or 57.5 and 57.3 (C-13); 54.9 (C-14 and C-17); 45.3 (C-7); 38.6 (C-11); 33.1 or 31.0 and 30.7 (C-12); 28.9 (C-20, C-21, C-22); 27.2 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₂₂H₃₄N₃O₄ ⁺, 404.2544; found, 404.2548.

3.1.8. General Methodology for the Synthesis of Substances 3a–3f

To a 50 mL round-bottom flask was added the respective amide (2a to 2f) and dichloromethane. The solution was kept under magnetic stirring and ice bath until 0 °C, and then, 2 mL of trifluoroacetic acid was added. The reaction was kept under these conditions and monitored by TLC (eluent: ethyl acetate; stain: iodine) until evidence of consumption of the starting material. Then, trifluoroacetic acid was first eliminated by blowing compressed air over the reaction mixture. The remaining material was then dissolved in dichloromethane (15 mL) and transferred to a separatory funnel. The organic phase was sequentially washed with distilled water (10 mL), 0.5 mol L^–1 of aqueous NaOH solution (25 mL), a saturated aqueous NH₄Cl solution (10 mL), and finally distilled water (10 mL). After drying over anhydrous Na₂SO₄, the solution was filtered and concentrated under reduced pressure to afford the desired products.

3.1.9. (S)–N-(Furan-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3a)

According to the general methodology described in 3.1.8, the reaction was conducted using 400 mg of the amide 2a, 7 mL of dichloromethane, and kept under magnetic stirring at 0 °C for 2 h after the addition of trifluoroacetic acid to obtain 244 mg (85% yield) of 3a as an orange solid. m.p.: degradation before melting; [α]_D (MeOH, 1% w/v): −70°; ¹H NMR (600 MHz, acetone-d ₆), δ/ppm: 7.44 (br s, 1H, H-15); 7.13–7.04 (m, 4H, H-1, H-2, H-3, and H-6); 6.34–6.33 (m, 1H, H-14); 6.23 (m, 1H, H-13); 4.41 (m, 2H, H-11); 4,03 (s, 2H, H-7, and H-8); 3.65 (dd, J _7,7’ e J _7,NH = 7.65 Hz, 1H, H-7); 3.08 (dd, J _8,9 = 7.2 Hz e J _9,9’ = 16.8 Hz, 1H, H-9); 2,87 (dd, J _8,9 = 9 Hz e J _9’,9 = 15.6 Hz, 1H, H-9′); 2.09 (s, 2H, NH and CONH); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 173.1 (C-10); 153.8 (C-12); 143.2 (C-15); 136.6 (C-4 or C-5); 135.4 (C-4 or C-5); 130.2–127.0 (C-1, C-2, C-3, and C-6); 111.6 (C-13 or C-14); 107.9 (C-13 or C-14); 57.4 (C-8); 47.8 (C-7); 36.8 (C-11); 32.0 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₁₅H₁₇N₂O₂ ⁺, 257.1285; found, 257.1285.

3.1.10. (R)-N-(Furan-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3b)

According to the general methodology described in 3.1.8, the reaction was conducted using 400 mg of the amide 2b, 6 mL of dichloromethane, and kept under magnetic stirring at 0 °C for 2 h after the addition of trifluoroacetic acid to obtain 253 mg (88% yield) of 3b as an orange solid. M.P.: degradation before melting; [α]_D (MeOH, 1% w/v): +80°; ¹H NMR (600 MHz, acetone-d ₆), δ/ppm: 7.44 (br s, 1H, H-15); 7.11–7.01 (m, 4H, H-1, H-2, H-3, and H-6); 6,34 (dd, J _13,14 = 2.1 Hz e J _14,15 = 1.2 Hz, 1H, H-14); 6.22 (d, J _13,14 = 3 Hz, 1H, H-13); 4.41 (d, J _CONH,11 = 4.2 Hz, 2H, H-11); 3.96 (s, 2H, H-7, and H-8); 3.52 (dd, J _7,7 = 5.4 Hz and J _7,NH = 4.8 Hz, 1H, H-7); 3.04 (dd, J _8,9 = 4.8 Hz and J _9,9 = 11.4 Hz, 1H, H-9); 2.81 (dd, J _8,9 = 6 Hz and J _9,9 = 10.2 Hz, 1H, H-9); 2.08 (s, 1H, CONH); 2.07–2.06 (m, 1H, NH); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 173.2 (C-10); 153.6 (C-12); 142.8 (C-15); 137.2 (C-4); 135.5 (C-5); 129.9–126.6 (C-1, C-2, C-3, and C-6); 111.2 (C-14); 107.5 (C-13); 57.3 (C-8); 47.9 (C-7); 36.4 (C-11); 31,8 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₁₅H₁₇N₂O₂ ⁺, 257.1285; found, 257.1283.

3.1.11. (S)–N-Benzyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3c)

According to the general methodology described in 3.1.8, the reaction was conducted using 671 mg of amide 2c, 10 mL of dichloromethane, and kept under magnetic stirring at 0 °C for 4 h after the addition of 2.5 mL of trifluoroacetic acid. After the workup described in 3.1.8, the combined aqueous layers were extracted with dichloromethane (3 × 15 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to yield 481 mg of crude material. A portion of this material (250 mg) was subsequently dissolved in dichloromethane (15 mL) and placed in a separatory funnel, where it was subjected to successive washes with distilled water (3 × 20 mL) followed by 1 mol L^–1 of aqueous HCl (2 × 15 mL). The resulting aqueous layers were combined and then alkalinized with aqueous solution of NaOH 1 mol/L until pH 12 and then extracted with dichloromethane (5 × 20 mL). The organic layers obtained from this step were again combined and dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under reduced pressure, obtaining 214 mg (44% yield) of 3c as a beige solid. M.P.: 100.5–103.5 °C; [α]_D (MeOH, 1% w/v): −72°; ¹H NMR (600 MHz, acetone-d ₆), δ/ppm: 7.31–7.28 (m, 4H, H-2, H-3, H-14, and H-16); 7.24–7.21 (m, 1H, H-15); 7.14–7.10 (m, 3H, H-1 or H-6, H-13, and H-17); 7.04–7.02 (m, 1H, H-1, or H-6); 4.46–4.40 (m, 2H, H-11); 3.97 (s, 2H, H-7, and H-8); 3.56 (dd, J _7,7 = 5.4 Hz and J _7,NH = 4.8 Hz, 1H, H-7); 3.06 (dd, J _8,9 = 4.8 Hz and J _9,9 = 11.3 Hz, 1H, H-9); 2.85 (dd, J _8,9 = 6.1 Hz e J _9,9 = 11.3 Hz, 1H, H-9); 2.08–2.06 (m, 1H, NH); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 173.4 (C-10); 140.7 (C-4, C-5 or C-12); 137.3 (C-4, C-5, or C-12); 135.6 (C-4, C-5, or C-12); 129.9–126.6 (C-1, C-2, C-3, C6, C-13, C-14, C-15, C-16, and C-17); 57.4 (C-8); 47.9 (C-7); 43.2 and 43.1 (C-11); 32.0 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₁₇H₁₉N₂O⁺, 267.1492; found, 267.1493.

3.1.12. (S)–N-(3-Phenylpropyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3d)

According to the general methodology described in 3.1.8, the reaction was conducted using 270 mg of amide 2d, 6 mL of dichloromethane and kept under magnetic stirring at 0 °C for 2 h after the addition of trifluoroacetic acid, obtaining 179 mg (89% yield) of 3d as an orange pasty solid. [α]^D (MeOH, 1% w/v): −60°; ¹H NMR (400 MHz, acetone-d ₆), δ/ppm: 7.25–7.10 (m, 9H, H-1, H-2, H-3, H-6, H-15, H-16, H-17, H-18, and H-19); 3.97 (s, 1.5H, H-8, and H-7); 3.54 (t, J _8,9 = 7 Hz, 0.5H, H-8); 3.47 (dd, J _7,7 = 5.2 Hz and J _7,NH = 4.8 Hz, 1H, H-7); 3.27 (td, J _11,12 = 10.5 Hz e J _11,CONH = 5.4 Hz, 2H, H-11); 3.03 (dd, J _8,9 = 4.8 Hz e J _9,9 = 11.6 Hz, 1H, H-9); 2.80 (dd, J _8,9 = 6 Hz e J _9,9 = 10.4 Hz, 1H, H-9); 2.64 (t, J _12,13 = 7.6 Hz, 2H, H-13); 2.09–2.07 (m, 2H, NH, and CONH); 1.83 (qt, J _12,13 and J _12,13 = 7.4 Hz, 2H, H-12); ¹³C NMR (150 MHz, acetone-d ₆), δ/ppm: 173.4 (C-10); 143.0 (C-14); 137.2 (C-4); 135.6 (C-5); 132.6–126.6 (C-1, C-2, C-3, C-6, C-15, C-16, C-17, C-18, C-19); 57.4 (C-8); 48.0 (C-7); 39.2 (C-11); 33.9 (C-9); 32.4 (C-13); 32,1 (C-12); HRMS (m/z) [M + H]⁺ calcd for C₁₉H₂₃N₂O⁺, 295.1805; found, 295.1808.

3.1.13. (S)-(4-Methylpiperazin-1-yl)­(1,2,3,4-tetrahydroisoquinolin-3-yl)­methanone (3e)

According to the general methodology described in 3.1.8, the reaction was conducted using 850 mg of amide 2e, 10 mL of dichloromethane and kept under magnetic stirring at 0 °C for 2 h after the addition of 2.5 mL of trifluoroacetic acid, obtaining 511 mg (83% yield) of 3e as an orange pasty solid. [α]_D (MeOH, 1% w/v): −52°; ¹H NMR (400 MHz, acetone-d ₆), δ/ppm: 7.13–7.03 (m, 4H, H-1, H-2, H-3, and H-6); 4.06 (m, 3H, H-7, and H-8); 3.72–3.54 (m, 4H, H-11, and H-14); 2.91 (dd, J _8,9 = 6 Hz and J _9,9 = 10.4 Hz, 1H, H-9); 2.82 (dd, J _8,9 = 5 Hz and J _9,9 = 11.6 Hz, 1H, H-9); 2.45–2.36 (m, 4H, H-12, and H-13); 2.26 (s, 3H, H-15); 2.09–2.07 (m, 1H, NH); ¹³C NMR (100 MHz, acetone-d ₆), δ/ppm: 130.0–126.6 (C-1, C-2, C-3, and C-6); 56.1 and 55.4 (C-12 and C-13); 53.6 (C-8); 47.4 (C-7, C-11 or C-14); 46.2 (C-15); 46.0 (C-7, C-11 or C-14); 42.4 (C-7, C-11, or C-14); 31.7 (C-9).

3.1.14. (S)–N-(3-Morpholinopropyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3f)

According to the general methodology described in 3.1.8, the reaction was conducted using 650 mg of amide 2f, 10 mL of dichloromethane and kept under magnetic stirring at 0 °C for 5 h after the addition of 2.5 mL of trifluoroacetic acid, obtaining 212 mg (43% yield) of 3f as an orange pasty solid. [α]_D (MeOH, 1% w/v): −60°; ¹H NMR (400 MHz, acetone-d ₆), δ/ppm: 7.13–7.03 (m, 4H, H-1, H-2, H-3, and H-6); 3.97–3.72 (m, 2H, H-7, and H-8); 3.65–3.63 (t, J _14,15 and J _16,17 = 4.6 Hz, 4H, H-15, and H-16); 3.45 (dd, J _7,7 = 5.2 Hz and J _7,NH = 4.8 Hz, 1H, H-7); 3.29 (t, J _11,12 = 6.6 Hz, 2H, H-11); 3.05 (dd, J _9,9 = 11.6 Hz and J _9,8 = 4.8 Hz, 1H, H-9); 2.78 (dd, J _8,9 = 6 Hz e J _9,9 = 10.2 Hz, 1H, H-9); 2.38 (m, 6H, H-13, H-14, and H-17); 1.67 (qt, J _11,12 e J _12,13 = 6.7 Hz, 2H, H-12); ¹³C NMR (100 MHz, acetone-d ₆), δ/ppm: 137.2 (C-4); 135.6 (C-5); 129.9–126.6 (C-1, C-2, C-3, and C-6); 67.4 (C-15 and C-16); 57.9 (C-13); 57.4 (C-8); 54.7 (C-14 and C-17); 48.0 (C-7); 38.6 (C-11); 32.0 (C-9); 26.6 (C-12); HRMS (m/z) [M + H]⁺ calcd for C₁₇H₂₆N₃O₂ ⁺, 304.2020; found, 304.2019.

3.1.15. General Methodology for the Synthesis of Hydrochlorides 4a, 4c, and 4d

The respective amide was solubilized in 15 mL of AcOEt, transferred to a separation funnel, and extracted with 3 × 20 mL of HCl 0.1 mol/L. The aqueous layers were combined and then dried over compressed air flow.

3.1.16. (S)-3-((Furan-2-ylmethyl)­carbamoyl)-1,2,3,4-tetrahydroisoquinolin-2-ium Chloride (4a)

According to the general methodology described in 3.1.15, the reaction was conducted using 51 mg of amide 3a, obtaining 56 mg (96% yield) of 4a as a brownish solid. m.p.: degradation before melting; [α]_D (MeOH, 1% w/v): −90°; ¹H NMR (600 MHz, DMSO-d ₆), δ/ppm: 10.13 (br s, 1H, NH ₂); 9.63 (br s, 1H, NH ₂); 9.36 (t, J _CONH,11 = 5.4 Hz, 1H, CONH); 7.60–7.58 (m, 1H, H-15); 7.24 (s, 4H, H-1, H-2, H-3, and H-6); 6.41 (br s, 1H, H-13, or H-14); 6.34 (br s, 1H, H-13, or H-14); 4.38–4.20 (m, 5H, H-7, H-8, and H-11); 3.38–3.34 (m, 1H, H-9); 3.02 (dd, J _9,9 = 12 Hz and J _9,8 = 4 Hz, 1H, H-9); ¹³C NMR (150 MHz, DMSO-d ₆), δ/ppm: 167.7 (C-10); 151.3 (C-12); 142.3 (C-15); 131.0 (C-5); 128.6 (C-3); 128.5 (C-4); 127.4 (C-6); 126.8 (C-1 or C-2); 126.5 (C-1 or C-2); 110.5 (C-13 or C-14); 107.2 (C-13 or C-14); 53.8 (C-8); 43.7 (C-7); 35.6 (C-11); 29.3 (C-9).

3.1.17. (R)-3-((Furan-2-ylmethyl)­carbamoyl)-1,2,3,4-tetrahydroisoquinolin-2-ium Chloride (4b)

To a 50 mL round-bottom flask containing 74 mg of 3b solubilized in 10 mL of acetone was added, under ice bath, 1 mL of 1.0 mol/L HCl. The mixture was kept under magnetic stirring for 5 min and then dried over compressed air flow, obtaining 68 mg (81% yield) of 4b as a brownish solid. M.P.: degradation before melting; [α]_D (MeOH, 1% w/v): +76°; ¹H NMR (600 MHz, DMSO-d ₆), δ/ppm: 10.11 (s, 1H, NH ₂); 9.62 (s, 1H, NH ₂); 9.35 (t, J _CONH,11 = 5.4 Hz, 1H, CONH); 7.61 (m, 1H, H-15); 7.27–7.22 (m, 4H, H-1, H-2, H-3, H-6); 6.42 (dd, J _13,14 = 1.8 Hz and J _14,15 = 1.2 Hz, 1H, H-14); 6.34 (m, 1H, H-13); 4.38 (t, J _7,NH = 5.4 Hz, 2H, H-11); 4.33–4.26 (m, 2H, H-7); 4.23–4.18 (m, 1H, H-8); 3.36 (dd, J _8,9 = 4.2 Hz e J _9,9 = 12.6 Hz, 1H, H-9); 3.02 (dd, J _8,9 = 4.8 Hz e J _9,9 = 12 Hz, 1H, H-9); ¹³C NMR (150 MHz, DMSO-d ₆), δ/ppm: 167.8 (C-10); 151,4 (C-12); 142.4 (C-15); 131.0 (C-4 and C-5); 128.6–126.6 (C-1, C-2, C-3 and C-6); 110.5 (C-13 or C-14); 107.3 (C-13 or C-14); 53.9 (C-8); 43.8 (C-7); 35.7 (C-11); 29.4 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₁₅H₁₇N₂O₂ ⁺, 257.1285; found, 257.1287.

3.1.18. (S)-3-(Benzylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-2-ium Chloride (4c)

According to the general methodology described in 3.1.15, the reaction was conducted using 100 mg of amide 3c, obtaining 112 mg (98% yield) of 4c as a beige solid. m.p.: 184.9–188,9 °C; [α]_D (MeOH, 1% w/v): −92°; ¹H NMR (600 MHz, DMSO-d ₆), δ/ppm: 10.04 (br s, 1H, NH ₂); 9.57 (br s, 1H, NH ₂); 9.40 (t, J _CONH,11 = 5.7 Hz, 1H, CONH); 7.35–7.25 (m, 9H, H-1, H-2, H-3, H-6, H-13, H-14, H-15, H-16, and H-17); 4.38–4.23 (m, 5H, H-7, H-8, and H-11); 3.39 (dd, J _8,9 = 4.2 Hz and J _9,9 = 12.6 Hz, 1H, H-9); 3.06 (dd, J _8,9 = 4.2 Hz and J _9,9 = 12 Hz, 1H, H-9); ¹³C NMR (150 MHz, DMSO-d ₆), δ/ppm: 167.9 (C-10); 138.6 (C-12); 131.0 (C-4 and C-5); 128.7–126.6 (C-1, C-2, C-3, C-6, C-13, C-14, C-15, C-16, and C-17); 54.0 (C-8); 43.9 (C-7 or C-11); 42.2 (C-7 or C-11); 29.4 (C-9); HRMS (m/z) [M + H]⁺ calcd for C₁₇H₁₉N₂O⁺, 267.1492; found, 257.1493.

3.1.19. (S)-3-((3-Phenylpropyl)­carbamoyl)-1,2,3,4-tetrahydroisoquinolin-2-ium Chloride (4d)

According to the general methodology described in 3.1.15, the reaction was conducted using 109 mg of amide 3d, obtaining 122 mg (quantitative yield) of 4d as a white solid. m.p.: degradation before melting; [α]_D (MeOH, 1% w/v): −90°; ¹H NMR (600 MHz, DMSO-d ₆), δ/ppm: 10.10 (br s, 1H, NH ₂); 9.54 (br s, 1H, NH ₂); 9.00 (t, J _CONH,11 = 5.4 Hz, 1H, CONH); 7.30–7.17 (m, 9H, H-1, H-2, H-3, H-6, H-15, H-16, H-17, H-18, and H-19); 4.34–4.17 (m, 3H, H-7, and H-8); 3.36 (dd, J _8,9 = 4.2 Hz and J _9,9 = 12.6 Hz, 1H, H-9); 3.18–3.16 (m, 2H, H-11); 3.02 (dd, J _8,9 = 4.2 Hz and J _9,9 = 12 Hz, 1H, H-9); 2.63 (t, J _12,13 = 7.5 Hz, 2H, H-13); 1.77 (qt, J _11,12 and J _12,13 = 7.2 Hz, 2H, H-12); ¹³C NMR (150 MHz, DMSO-d ₆), δ/ppm: 167.6 (C-10); 141.6 (C-14); 131.1 (C-4 or C-5); 128.6–125.8 (C-1, C-2, C-3, C-6, C-15, C-16, C-17, C-18, and C-19); 53.9 (C-8); 43.7 (C-7); 38.3 (C-11); 32.4 (C-9); 30.7 (C-12 or C-13); 29.4 (C-12 or C-13).

3.2. In Silico Studies

We analyzed the properties of THIQA derivatives using a computational workflow designed to compare the relevant conformations of those compounds. Initially, conformational analysis was performed using the SPARTAN’20 V1.1.4 software, employing the MMFF94s force field to generate a diverse set of low-energy conformers. All conformers generated in this step were retained for further analysis to capture the potential conformational flexibility. Each of these conformers is then subjected to geometry optimization using the semiempirical PM6 method Then, another geometry optimization was carried out using DFT at the B3LYP functional − with the 6-31G* basis set. ,

3.3. Biological Assays

3.3.1. Cruzain Inhibition Assay

Recombinant cruzain was kindly provided by Allison Doak and Brian Shoichet (University of California, San Francisco, CA, USA). Cruzain inhibition assays were performed according to previously reported procedures. , Briefly, enzymatic activity was monitored through the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC using recombinant cruzain in sodium acetate buffer (pH 5.5) supplemented with β-mercaptoethanol and Triton X-100. Fluorescence was recorded over time at appropriate excitation (340 nm) and emission (440 nm) wavelengths with DMSO and E-64 employed as negative and positive controls, respectively. Compounds were initially screened at 100 μM, and inhibition percentages were calculated from initial reaction rates in the presence of compounds to those observed for the DMSO control. IC₅₀ values were obtained by nonlinear regression analysis, with all measurements performed in triplicate in independent experiments.

3.3.2. In Vitro Activity against T. cruzi Intracellular Amastigotes and Early Trypomastigotes

The in vitro anti-T. cruzi activity was evaluated on L929 mouse fibroblasts infected with Tulahuen strain of the T. cruzi expressing the Escherichia coli β-galactosidase as a reporter gene, following the procedure described previously. This assay format allows for the assessment of compound activity across the intracellular developmental cycle of the parasite, including the early trypomastigote stage and the replicative amastigote stage. Briefly, for the bioassay, 4000 L929 cells were added to each well of a 96-well microtiter plate. After an overnight incubation, 40,000 trypomastigotes were added to the cells and incubated for 2 h. Then, the medium containing extracellular parasites was replaced with 150 μL of fresh medium, and the plate was incubated for an additional 48 h to establish the infection. During this period, internalized trypomastigotes naturally differentiate into amastigotes, while extracellular parasites are eliminated; thus, both early intracellular trypomastigotes and amastigotes are present within host cells. For IC₅₀ determination, the cells were exposed to each synthesized compound at serial decreasing dilutions, and the plate was incubated for 96 h. This extended exposure period covers the full intracellular cycle of T. cruzi, enabling phenotypic evaluation of the compound activity against both parasite stages. After this period, 50 μL of 500 μM chlorophenol red beta-d-galactopyranoside (CPRG) in 0.5% Nonidet P40 was added to each well, and the plate was incubated for 16–20 h, after which the absorbance at 570 nm was measured. Controls with uninfected cells, untreated infected cells, infected cells treated with benznidazole at 3.8 μM (1 μg/mLpositive control), or DMSO 1% were used. The results were expressed as the percentage of T. cruzi growth inhibition in compound tested cells compared to that of the infected cells and untreated cells. The IC₅₀ values were calculated by linear interpolation. Quadruplicates were run on the same plate, and the experiments were repeated at least once.

3.3.3. In Vitro Cytotoxic Activity Test of Compounds and CC₅₀ Determination over L929 Cell Line

For this assay, L929 fibroblasts (4000 cells per well) were seeded in 96-well microplates containing 150 μL of RPMI-1640 medium (pH 7.2–7.4; Gibco BRL) supplemented with 10% fetal bovine serum and 2 mM glutamine and incubated at 37 °C for 3 days. The culture medium was subsequently replaced, and the cells were treated with increasing concentrations of the test compounds, starting at the IC₅₀ value determined for T. cruzi. Following 96 h of exposure, alamarBlue was added, and absorbance was measured at 570 and 600 nm after 4–6 h. Untreated cells and cells exposed to 1% DMSO were included as controls. Cell viability was calculated based on the differential reduction of alamarBlue between treated and control wells, and the concentration required to reduce cell viability by 50% (CC₅₀) was determined. Quadruplicates were run in the same plate, and the experiments were repeated at least once.

IC₅₀ over T. cruzi and L929 cells were determined by linear interpolation, and the selectivity index (Supporting Information) was calculated by the ratio of CC₅₀ L929 cells/IC₅₀ T. cruzi.

4. Conclusion

In the present study, we synthesized and characterized six final amide derivatives of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (3a–f) and four hydrochlorides (4a–d). The compounds were subjected to enzymatic and cellular assays to evaluate their activity against cruzain and T. cruzi. Among the tested molecules, 4a and 4b displayed the highest cruzain inhibition, and their IC₅₀ values were determined. However, both compounds exhibited low potency when compared to commonly applied hit-identification (IC₅₀ < 5 μM). In cellular assays, 3d and 4d showed the best activity against T. cruzi, with IC₅₀ values comparable to those of the positive control benznidazole. Furthermore, the activity of these substances against T. cruzi appears to be independent of cruzain inhibition since their inhibition percentages were low. In light of the modest activity observed against cruzain, structural optimization is required in an effort to obtain more potent inhibitors. Accordingly, additional modificationssuch as alterations to the tetrahydroisoquinoline ring, introduction of strategic substituents, and optimization of the side chainmay further enhance the potency of these 1,2,3,4-tetrahydroisoquinoline amide derivatives toward this enzyme.

Moreover, substance 3d showed excellent results against T. cruzi and may be a candidate for further testing, such as in vivo tests. Furthermore, studies can be carried out to investigate by which mechanism this substance inhibits parasite growth.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11033.

• Spectroscopic characterization of synthesized compounds (NMR and HRMS) (PDF)

The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brazil (ROR identifier: 00x0ma614).

The authors declare no competing financial interest.