Instrumentation

All reactions requiring inert atmosphere were carried out under nitrogen atmosphere. Drying of solvents was achieved using molecular sieve for 48 h. All reagents were purchased from commercial suppliers, Aldrich, Merck, Fluka, Avra, SD fine and Alfa Aesar. Thin layer chromatography was carried out using silica plates purchased from Avra. The plates were visualized under UV light (popular India). FT-IR spectroscopy of the compounds were run in PerkinElmer Spectrum version 10.03.06 and the bands presented in wavenumber. Proton and carbon-13 NMR spectroscopy were run in DMSOd₆ and CD₃OD, unless otherwise stated on either Jeol 500 MHz or 400 MHz. The chemical shifts were reported in part per million with reference to tetramethylsilane. Mass spectroscopy were carried out using micro TOF electrospray time of flight (ESI-TOF) mass spectrometer (Aerodyne Research Inc. USA), sodium formate was used as the calibrant. All experiments were carried out at Prof. Sandeep Verma’s Laboratory, department of Chemistry, Indian Institute of Technology, Kanpur. Melting points were determined using digital melting point apparatus (Stuart, SMP20) and were uncorrected.

General procedure for the synthesis of substituted benzenesulphonamoyl alkanamides (7a-x)

Sodium carbonate (Na₂CO₃, 1.590 g, 15 mmol) was added to a solution of amino acids (6a-h, 12.5 mmol) in water (15 mL) with continuous stirring until all the solutes dissolved. The solution was cooled to -5°C and an appropriate benzenesulphonyl chloride (5a-c, 15 mmol) was added in four portions over a period of 1 h. The slurry was further stirred at room temperature for 4 h. The progress of the reaction was monitored by using TLC (MeOH/DCM, 1:9). Upon completion, the mixture was acidified using 20% aqueous hydrochloric acid to pH 2. The crystals was filtered via suction and washed with pH 2.2 buffer. The pure products (7a-x) were dried over self-indicating fused silica gel in a desiccator.

General procedure for the synthesis of N-benzoyl derivatives of benzenesulphonamides (9a-f, i-n and q-v)

Appropriate benzenesulphonamides (7a-f, i-n and q-v, 1.0 mmol) was dissolved in NaOH (10%, 10 mL) in a 50 mL round bottom flask and benzoyl chloride (8, 1.1 mmol, 0.2 mL) was added into the solution and stirred at room temperature. The reaction progress was monitored by TLC (3% MeOH/DCM) to the disappearance of the benzenesulphonamide spot. Upon completion, the solution was transferred into a beaker containing crushed ice and then acidified to pH 3 with concentrated hydrochloric acid. The solid was collected via suction filtration and transferred into a beaker containing CCl₄ (10 mL) and covered with watch glass boiled for 10 min. The mixture was allowed to cool slightly and then filtered. The products (9a-f, i-n and q-v) obtained were washed with 10–20 mL CCl₄ and dried over fused self-indicating silica gel in a desiccator.

Boric acid catalysed direct amidation of unactivated carboxylic acid and 3-aminoquinoline

To a suspension of N-benzoyl-substituted-benzenesulphonamides (9a-f, i-n, q-v, 7g-h, o-p and w-x) (1.0 mmol) (1.0 mmol) in dry toluene (40 mL) equipped with Dean-Stark apparatus for azeotropic removal of water, was added 3-aminoquinoline (1.0 mmol) and boric acid (0.1 mmol) at room temperature and refluxed for 8 h. Upon completion (as monitored by TLC), the amide products were precipitated out from the reaction mixture by adding 40 mL n-hexane. The carboxamides were obtained via suction filtration, washed with n-hexane and dried over fused silica gel or concentrated using rotary evaporator and dried over vacuum in the case of oily products.

Pharmacokinetic prediction

The drug-likeness was predicted using online quantitative estimation of drug-likeness tools. The prediction was based on the calculated logP, topological polar surface area (TPSA), hydrogen bond donors (HBD), hydrogen bond acceptor (HBA), number of acid (NA), molecular weight (MW), number of rotatable bonds (NRB) and percentage absorption (ABS). The prediction tool was accessed from crdd.osdd.net/oscadd/qed/submit_virtual.php, molinspiration and druglito.

In vitro antitrypanosomal activities

In vitro antitrypanosomal activities against T. b. gambiense strain was described by Otoguro et al [25]. In brief, T. b. gambiense strain was cultured in IMDM with various supplements and 10% heat-inactivated FBS at 37°C, under 5.0% CO₂/95% air according to the method of Baltz et al [26]. Ninety five mL of the trypanosomes suspension (2.0–2.5104 trypanosomes/mL for strain GUTat 3.1) was seeded in a 96-well microplate, and 5.0 mL of a test compound solution (dissolved in 5.0% dimethylsulfoxide) was added followed by incubation for 72 h (long incubation-low inoculation test: LILIT). Ten mL of the fluorescent dye Alamar Blue was added to each well. After incubation for 3–6 h, the resulting solution was read at 528/20 nm excitation wavelength and 590/35 nm emission wavelength by a FLx800 fluorescent plate reader (Bio-Tek Instrument, Inc. Vermont, USA). Data were transferred into a spreadsheet program (Excel). The IC₅₀ values were determined using fluorescent plate reader software (KC-4, Bio-Tek). Successive subcultures were done in 24-well tissue culture plates under the same conditions.

Determination of LD₅₀ for the active anti-inflammatory compounds

Male mice were divided into various groups and test compounds were administered in various doses intraperitoneally. Following treatments, the animals were observed for up to 4 h continuously and were then kept under observation for 72 h. All behavioral changes and deaths during the observation periods were recorded. The percentage of death at each dose level was then calculated, converted to probits and the LD₅₀ (μM/kg) values were calculated [27]. To observe the health status of the mice, they were monitored 4 times a day. Humane endpoints were used when the animal shows sign of weight loss, weakness accompanied by inability to get food, complete anorexia and convulsion for 24 h. For the purpose of ameliorating the suffering of the dying mice, CO₂ euthanasia was applied. All the dead mice were disposed in bio-safety containers in accordance with local standard protocols. The mortality rate in each group was calculated according to the formula:

In vitro antitrypanosomal activities and structure-activity relationship

The results of the in vitro antitrypanosomal activities (Fig 4), revealed that compounds 11f, 11g, 11h, 11n, 11o, 11p, 11v, 11w and 11x had IC₅₀ values of 3, 2, 5, 1, 3, 4, 1, 2, 3 nM respectively which in exception of compounds 11h and 11p were 2-fold, 3-fold, 5-fold, 2-fold, 5-fold, 3-fold and 2-fold more active than melarsoprol (IC₅₀ 5 nM). These derivatives with good antitrypanosomal activity against T. b. gambiense could serve either as a replacement to melarsoprol and pentamidine where there is record of toxicity or as a new entrant to HAT chemotherapy. The L-valine, L-hydroxyproline and L-proline derivatives were the most active compounds in each of the series. The 4-nitrobenzenesulphonamide derivatives were found to be more active than the unsubstituted benzenesulphonamide and 4-methylbenzenesulphonamide derivatives. The p-nitrobenzenesulphonamides were the most active derivatives followed by the p-toluenesulphonamides and then the benzenesulphonamides. These findings underscores the importance of a substituent at the para position of the benzene ring in the antitrypanosomal activity. In general, the effect of the substituents on the benzene ring followed the trend NO₂>CH₃>H. The effect of the amino acid residue showed that the L-valine derivatives (11f, 11n and 11v) had better activity than the L-leucine (11d, 11l and 11t), L-isoleucine (11e, 11m and 11u) and glycine (11a, 11i and 11q) derivatives. This shows that the chain length of a substituent at the β-carbon contributes significantly to biological activity. The effect of the presence of a substituent at the β-carbon was shown by the biological activity of the L-leucine, L-isoleucine and L-valine derivatives which were better than the glycine derivatives that lacked substitution at the β-carbon. Among the aromatic amino acids, the L-tryptophan (11c, 11k and 11s) were more active than the L-phenylalanine (11b, 11j and 11r), this may not be unconnected with the presence of nitrogen on the ring. The L-4-hydroxyproline (11g, 11o and 11w) were more active than their corresponding L-prolines (11h, 11p and 11x) which highlights the importance of the hydroxyl group on biological activity of the molecules.

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Fig 4. In vitro antitrypanosomal activities.

A table of value representing the in vitro antitrypanosomal activities.

https://doi.org/10.1371/journal.pone.0191234.g004

The LD₅₀ values showed that the most promising hits in exception of the p-nitrobenzenesulphonamide derivatives (11v-x) had better toxicity profile than melarsoprol and as such may likely not have the toxicity problems associated with melarsoprol and other antitrypanosomal agents currently in use. Compound 11h was the safest hit with LD₅₀ of 18.02 μM/kg. The p-toluenesulphonamide derivatives (11f-h) were the safest derivatives followed by the benzenesulphonamide derivatives (11n-p).

In silico calculation of the pharmacokinetics properties (ADME-T) and drug-likeness

Drug-likeness describes an integrated equilibrium between multiple molecular properties and structural features that define whether a particular compound is comparable to known drugs. Lipinski’s rule of 5 (ro5) is among the most common principle applied to evaluate drug-likeness properties of a compound [28]. Verber’s criteria have also been applied extensively to evaluate oral bioavailability of compounds [29]. These properties measure hydrophilicity, electronic distribution, hydrogen bonding capability, molecule size and flexibility that would affect molecules behaviours in a living system. The behavior predicted includes bioavailability, transport properties, affinity to proteins reactivity and toxicity [30].

A computational studies of all the carboxamides 11a-x using DrugLiTo and MedChem Designer 3.0 software was carried out to determine the Lipinski’s molecular properties, the blood-brain barrier likeness, and the number of rotatable bonds (NRB), together with the topological polar surface area (TPSA; a sum of polar atoms surfaces, a descriptor for drug absorption, penetrability and bioavailability) and the percentage of absorption (ABS %) calculated as (ABS % = 109–0.345×TPSA) [31].

The results of the pharmacokinetics prediction (Fig 5) revealed that all the tested compounds in exception of compound 11c complied with ro5 where LogP values ranged between -0.154 and 4.650 (≤ 5), MW range 381–619 (≤ 500 but one violation is permissible for ro5), HBA range 4–6 (≤10), HBD range 1–2 (≤5), suggesting that these compounds would not be expected to cause oral bioavailability problems. Compound 11c needs derivatization to improve on its oral bioavailability profile. In exception of compounds 11r-11u (NRB = 11), all the compounds showed NRB values range 5–10 (≤10), indicating acceptable molecular flexibility with consequent expected good permeability and oral bioavailability. Additionally, all the compounds evaluated showed TPSA range 75.19–135.40 Ų (≤140 Ų), showing good permeability and transport of the compounds in the cellular plasma membrane. Furthermore, all the tested compounds exhibited a considerable %ABS range 62.29–83.06% which is a designation of good bioavailability upon oral administration. Finally, since sleeping sickness occurs when single-cell trypanosome parasites spread from to brain over the blood-brain barrier, the BBB likeness of the compounds were evaluated. All the evaluated compounds showed NA values 0 (0), HBA range 4–6 (˂10) and MW range 381–619 (˂500), indicating that compounds 11b-11e, 11j-11k and 11r-11v cannot cross the blood-brain barrier and as such would not succeed in the treatment of second stage of human African trypanosomiasis. It is important to note that the more active derivatives in the in vitro antitrypanosomal activities study (11f-h, 11n-p and 11v-x) had fascinating pharmacokinetics properties.

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Fig 5. Pharmacokinetic properties in vitro antitrypanosomal activities.

A table of value repreenting the in vitro antitrypanosomal activities.

https://doi.org/10.1371/journal.pone.0191234.g005