RESEARCH ARTICLE

 

Synthesis, in vitro Cytotoxicity and Trypanocidal Evaluation of Novel 1,3,6-Substituted Non-fluoroquinolones

 

 

Richard M. Beteck^{I, *}; Michelle Isaacs^III; Heinrich C. Hoppe^{II, III}; Setshaba D. Khanye^{I, III, IV, *}

^IDepartment of Chemistry, Rhodes University, Grahamstown, 6140, South Africa
^IIDepartment of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa
^IIICentre for Chemico- and Biomedical Research, Rhodes University, Grahamstown, 6140, South Africa
^IVFaculty of Pharmacy, Rhodes University, Grahamstown, 6140, South Africa

 

 

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ABSTRACT

Sleeping sickness (trypanosomiasis) is a neglected tropical disease that affects mostly the poorest communities in sub-Saharan Africa. Toxic side effects associated with the use of current anti-trypanosomal drugs, which in some cases kill faster than the disease itself, necessitate the search for new drugs with better safety margins. To this effect, a small library bearing different substituents at position -1, -3, and -6 of the quinolone nucleus were synthesized and evaluated in vitro against HeLa cell lines and Trypanosoma brucei brucei for cytotoxicity and trypanocidal potentials, respectively. While most of these compounds showed no cytotoxic effect, they exhibited moderate to weak anti-trypanosomal activities. The SAR studies of this series provide new information worth considering in future exploration of the quinolone scaffold in search of more potent and safe trypanocidal agents.

Keywords: Sleeping sickness, trypanosomiasis, quinolones, non-fluoroquinolones.

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1. Introduction

Human African trypanosomiasis (HAT), also referred to as sleeping sickness,¹ is among the WHO's list of neglected tropical diseases (NTDs).² It mostly affects people living in rural areas of sub-Saharan Africa,³ where medical facilities are scarce and drug purchasing power of inhabitants is very low.⁴ At least 2184 new cases of the disease were reported in 2016,⁵ and approximately 60 million people living in 36 different countries are presently at risk of contracting the disease.⁶

HAT takes two forms caused by two different subspecies of Trypanosoma brucei.⁷ T. ". gambiense is the pathogenic subspecies causing the form of HAT commonly found in central and western Africa,^8,9 whereas T. ". rhodesiense is the subspecies responsible for the form of HAT prevalent in eastern and southern Africa.¹⁰ It has been noted that the two sub-pathogenic species coexist in Uganda.¹¹ These pathogens are transmitted between humans following the bite of an infected tsetse fly.¹² The disease exists in two stages:^13,14 haemolymphatic stage - wherein the parasites are localized in blood and lymphatic systems,¹⁵ and an encephalitic stage - wherein the parasites have invaded the central nervous system.^16,17

Current treatment options are limited to just four drugs.¹⁸ In any case of HAT, the drug to be used is dictated by the form and stage of the disease. Pentamidine is the drug of choice for treating the haemolymphatic stage of HAT caused by T.". gambiense,¹⁹ while a combination of nifurtimox and eflornithine is used to treat the encephalitic stage.²⁰ In cases of T ". rhodesiense, suramin is the recommended drug for treating the haemolymphatic stage,²¹ while malarsoprol is used to treat the encephalitic stage.²² Besides limited treatment options, the foregoing drugs are far from ideal. They all have poor oral bioavailability (and hence are administered intravenously), and considerable toxic side effects.²³ For example, pentamidine causes hyper or hypoglycae-mia and hypotension,²⁴ suramin causes renal failure, eflor-nithine causes alopecia and seizures,²⁵ while at least 5.9 % of patients on malarsoprol die from its toxicity,²⁶ creating a scenario wherein patients either die from an acute illness or die faster from a pill.

The overwhelming life-threatening side effects of existing drugs used to treat sleeping sickness create a dire need for new compounds with better drug properties such as high oral bioavailability, and a high safety margin. The therapeutic potentials of quinolones cannot be over emphasized. Compounds containing this scaffold are currently in use as drugs to treat bacterial and viral infections as well as other conditions such as cancers.²⁷ Fluoroquinolones (Fig. 1a) in clinical use have been extensively screened against trypanosomes and their activity profiles established as moderate to poor.^28,29,30 A hit optimization study by Hiltensperger and co-workers generated a potent library of fluoroquinolones characterized by benzylamides, (a)cyclic amines, and aliphatic chains at positions -3, -7, and -1 of the quinolone nucleus. However, the lead compound (Fig. 1b) in this series suffers from poor solubility,³¹ necessitating further work on this class of compounds. Unlike fluoroquinolones, the anti-trypanosomal potentials of non-fluorinated quinolones have not been extensively investigated, with just one study on non-fluorinated quinolones bearing substituents at position -1 and -2, respectively, being reported³² (Fig. 1c). To further expand the SAR around the non-fluoquinolone scaffold, we conceptualized and synthesized a library of 18 non-fluorinated quinolones bearing unique concurrent substituents at position -1, -3, and -6. Target compounds were subjected to in vitro cytotoxicity and anti-trypanosomal evaluation. At 20 μΜ concentration, most of the compounds exhibited less than 50 % parasite viability while having little effect on the viability of HeLa cell lines (see Supplementary information). This suggests that the quinolone scaffold can be tailored via chemical synthesis to generate safe and potent drug substrates to treat trypanosomiasis.

 

2. Results and Discussion

We conceptualized and synthesized a set of 18 novel compounds that bear different substituents at positions -1, -3 and -6 of the quinolone scaffold. Conceptualized compounds were synthesized as depicted in Scheme 1. Briefly, 4'-nitroaceto-phenone was reduced to 4'-aminoacetophenone using reduced iron powder and acetic acid.³³ 4'-Aminoacetophenone and 4-chloroaniline were each treated with diethyl ethoxy-methylenemalonate in refluxing acetonitrile to form condensed methylenemalonate esters, which underwent cyclization in boiling diphenylether at 245-250 °C for 5 min to form compound 2. Deprotonation of 2 using K₂CO₃, followed by N-alkylation with alkylhalides afforded target compounds 3a-h in yields ranging between 40 and 70 %. Ester functionality in compounds 3d-h underwent selective aminolysis in the presence of a ketone to afford target compounds 4a-j in 30-50 % yields. This transformation was realized using DBU as a base. All targeted compounds were characterized using proton and carbon NMR, HRMS and IR. The carbon NMR spectra of all compounds show a peak at c. 174 ppm, which is indicative of an oxo carbon within the quinolone nucleus (C-4) and the peak at c. 164 ppm is assigned to ester or amide carbonyl carbon (C-3a). With the exception of compounds 3a-c, the proton NMR spectra of all compounds show a singlet signal at ~2.3-2.6 ppm, which is assigned to a methyl attached to a carbonyl carbon (CH₃C=O). The carbonyl carbon is also evident in the respective carbon spectra at ~197ppm. The presence of a triplet peak (J = 5.9 Hz) at c. 9.9 ppm in the proton NMR spectra of compounds 4a-j, which is absent in compounds 3d-h, suggests the successful conversion of ester to amide. It is also worth noting that the acquisition of NMR spectroscopic data was at times hindered by compounds crystallizing out of the solutions. This sometimes necessitated the use of hot solvents to encourage compounds to remain in solution long enough to obtain 1H and 13C NMR spectroscopic data (see Supplementary information). The absorption band at 1654/cm on the IR spectrum further confirms the presence of amide.

This focused library was screened in vitro against human cervix adenocarcinoma (HeLa) cell lines to investigate potential cytotoxicity effects. The compounds were incubated at 20 μΜ in 96-well plates containing HeLa cells for 48 h. The numbers of cells surviving upon drug exposure were determined using resazurin reduction to resorufin by live cells and reading resorufin fluorescence in a multiwell plate reader. Compounds were tested in duplicate, and a standard deviation (S.D.) calculated. Results are expressed as % viability based on fluorescence readings in treated wells versus untreated control wells. Emetine (which induces cell apoptosis) was used as positive control. With the exception of compound 4f (% viability, -19 %), and 4j (% viability, -13 %), which strongly inhibited HeLa cell lines, the rest of the series had little to no effect on HeLa cell viability. This observation suggests that this series with necessary optimization could serve as templates for the development of non-toxic anti-trypanosomal agents.

The compounds were further evaluated in vitro for anti-trypanosomal activities by screening against the 427 strain of T.b. brucei. Compounds were added to in vitro cultures of T b. brucei in 96-well plates at a 20 μΜ concentration. After an incubation period of 48 h, the numbers of parasites surviving drug exposure were determined by adding resazurin. As in HeLa cells, resazurin is reduced to resorufin by living parasites. Resorufin is a fluorophore (Exc₅₆₀/Em₅₉₀) and can thus be quantified in a multiwell fluorescence plate reader. Compounds were tested in duplicate wells, and a standard deviation (S.D.) calculated. Results are expressed as % viability - the resorufin fluorescence in compound-treated wells relative to untreated controls. Pentamidine (an existing drug for treatment of trypanosomiasis) was used as a positive control. At 20 μΜ concentration, 11 compounds inhibited parasite growth below 50 % (Table 1); however, only compounds inhibiting parasite viability below 25 % with little or no effect on HeLa cell line were considered for IC₅₀ determination. The IC50 values for the selected compounds are summarized in Fig. 2.

 

 

Structure-activity relationship analysis across this series suggests that modifications at position -1, -3, and -6 of the quinolone scaffold influence anti-trypanosomal activities. For example, comparing the activity profiles of compounds 3b (IC₅₀, 19 μΜ) and 3g (IC₅₀, 24 μΜ), all having the same substituents at position -1 and -3 and differing in substitution pattern only at position -6 suggests that a chloride substituent at this position leads to increase anti-trypanosomal activity than a ketone. Also, comparing compounds 3d (IC₅₀,32 μΜ) bearing an alkyl chain at position -1 and 3g (IC₅₀, 24 μΜ) bearing a substituted-benzyl moiety at position -1, indicates that the presence of a substituted-benzyl moiety at position -1 seems to promote antitrypanosomal activities over alkyl chains. We also observed that the substituent on the benzyl moiety at position -1 also influences anti-trypanosomal activities. This is evident when comparing the effects of compounds 3a, 3b and 3c on parasite viability at a concentration of 20 μΜ. Compounds 3a and 3b bearing -NO₂, and -Br, respectively, exhibited more than 75 % parasite growth inhibition, while compound 3c having -CF₃ substituent exhibited less than 5 % parasite growth inhibition. These results suggest that electron-withdrawing units promote activity while electron-donating unit leads to poor activity. The substitution pattern at position -3 also seems to greatly influence activity. Comparing the activity of compounds 3g (ΚΖ₅₀, 24μΜ) and 4g (IC₅₀,7μΜ), both of which differ only in the substituent at position -3, suggests that an amide moiety at position -3 seems to enhance activity over ethyl ester.

 

3. Conclusion

We have synthesized a series of novel quinolones with varied substituents at position -1, -3 and -6 of the quinolone scaffold. While most compounds in this series showed no promising cytotoxicity potentials, compounds 4g emerged as potent anti-trypanosomal hit with IC₅₀ value of 7 μΜ. Although this series exhibited moderate to weak activities profiles, the comprehensive structure-activity relationship analyses of this series will undoubtedly serve as a resource for further optimization of the quinolone scaffold in search of new and potent anti-trypano-somal agents.

 

4. Experimental

4.1. General Method

All the chemicals and solvents used were purchased from various chemical suppliers and were used without further purification. Melting points were determined using a Reichert hot stage microscope and are uncorrected. The progress of the reactions was monitored by thin layer chromatography (TLC) using Merck F254 silica gel plates supported on aluminium. The crude products were purified by a silica gel column chromatog-raphy using Merck Kieselgel 60 A: 70-230 (0.068-0.2 mm) silica gel mesh. ¹H and ¹³C NMR spectra were recorded on Bruker Biospin 300 MHz, or 400 MHz spectrometers, and the chemical shifts are given in d values referenced to solvents and are reported in parts per million (ppm). The high-resolution mass spectrometric data of final compounds was recorded on a Waters Synapt G2 quadrupole time-of-flight (QTOF) mass spectrometer operated with an electrospray ionization probe in the positive mode (University of Stellenbosch). The instrument was operated with an electrospray ionization probe in the positive mode. The starting quinolones 2 were synthesized from the synthetically accessible compounds 1 as previously described in literature.³⁴

4.2. Synthesis of Compounds

4.2.1. General Method for the Preparation of N-alkylated Compounds 3a-h

A mixture of 2 (3.86 mmol, 1 g, 1 eq), K₂CO₃ (5.0 mmol, 0.53 g), alkyl halide (5 eq.) in acetone (50 mL) was refluxed for 15 h. Upon reaction completion as indicated by TLC, the mixture was filtered, and the filtrate evaporated to dryness to obtain a crude N-alkylated product which was purified through silica gel column chromatography using CH₂Cl₂/MeOH (10:1) as the mobile phase. Compounds 3a-h were obtained in 40-70 % yield following this procedure.

Methyl 6-chloro-1-(4-nitrobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate, 3a

Brown powder, 0.515 g (48 %), R = 0.81 (DCM/MeOH 10:1) m.p. 153-155 °C; ¹H NMR (300 MHz, DMSO) d 9.00 (s, 1H, H-2), 8.16 (d, J = 2.5 Hz, 1H, H-5), 7.89-7.57 (m, 3H, H-1c, H-8), 7.48 -7.05 (m, 3H, H-7, H-1d), 5.82 (s, 2H, H-1a), 4.24 (s, 3H, H-3c). ¹³C NMR (75 MHz, DMSO) d 172.2 (C-4), 164.1 (C-3a), 151.3 (C-2), 141.1 (C-3), 138.2 (C-6), 133.2 (C-1d), 131.3 (C-1c), 130.5 (C-5), 130.1 (C-7), 127.6 (C-1b), 126.3 (C-4a), 125.9 (C-8), 120.7 (C-8a), 110.9 (C-1e), 57.3 (C-3c), 55.7 (C-1a). IR (neat, cm^-1): 3093, 2972, 2931,1702,1686; ESI-HRMS m/z [M+H]+ calcd for C₁₈H₁₄ClN₂O₅ 373.0586, found 373.0594.

Ethyl 1-(4-bromobenzyl)-6-chloro-4-oxo-1,4-dihydroquinoline-3-carboxylate, 3b

White powder, 0.015 g (46 %), R = 0.8 (DCM/MeOH 10:1), m.p. 196-198 °C; ¹H NMR (400 MHz, DMSO) d 8.94 (s, 1H, H-2), 8.16 (d, J = 2.5 Hz, 1H, H-5), 7.81-7.48 (m, 4H, H-7, H-8, H-1c), 7.21 (d, J = 8.4 Hz, 2H, H-1d), 5.68 (s, 2H, H-1a), 4.25 (q, J = 7.1 Hz, 2H, H-3c), 1.30 (t, J = 7.1 Hz, 3H, H-3d). ¹³C NMR (101 MHz, DMSO) d 172.0 (C-4), 165.0 (C-3a), 151.0 (C-2), 138.5 (C-3), 135.5 (C-6), 132.9 (C-5), 132.2 (C-1d), 130.6 (C-1b), 129.9 (C-1c), 128.9 (C-4a), 125.9 (C-7), 121.8 (C-1e), 120.6 (C-8), 111.1 (C-8a), 60.3 (C-3c), 55.6 (C-1a), 14.2 (C-3d). IR (neat, cm^-1): 3096, 2978, 2941, 1702, 1680. ESI-HRMS m/z [M+H]+ calcd for C₁₉H₁₆BrClNO₃ 419.997, found 419.9998.

Ethyl 6-chloro-4-oxo-1-(4-(trifluoromethyl)benzyl)-1,4-dihydroquino-line-3-carboxylate, 3c

Brown powder, 0.5 g (70 %), R = 0.81 (DCM/MeOH 10:1), m.p. 203-205 °C; ¹H NMR (400 MHz, Pyridine) d 9.19 (s, 1H, H-2), 7.65 (d, J = 8.4 Hz, 2H, H-1c), 7.56-7.49 (m, 3H, H-5, H-7, H-8), 7.43 (d, J = 8.4 Hz, 2H, H-1d), 5.00 (s, 2H, H-1a), 4.36 (q, J = 6.9 Hz, 2H, H-3c), 1.23 (t, J = 6.9 Hz, 3H, H-3d).¹³C NMR (101 MHz, Pyridine) d 173.0 (C-4), 164.7 (C-3a), 150.64 (C-2), 140.6 (C-3), 140.2 (C-1b), 137.9 (C-6), 135.5 (C-1d), 132.9 (C-1c), 130.9 (C-5), 130.2 (C-7), 127.2 (C-1e), 126.4 (C-4a), 125.9 (C-8), 120.7 (C-8a), 119.5 (C-1f), 112.2 (C-1f), 60.3 (C-3c), 56.0 (C-1a), 14.2 (C-3d). IR (neat, cm^-1): 3003, 2962, 2921, 1705, 1684, ESI-HRMS m/z [M+H]+ calcd for C₂₀H₁₆ClF₃NO₃ 410.0765, found 410.0766.

Ethyl 6-acetyl-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate, 3d

Red powder, 0.65 g (70 %), R_f= 0.71 (DCM/MeOH 10:1); m.p. 143-147 °C; ¹H NMR (300 MHz, CDCl₃) d 8.97 (s, 1H, H-2), 8.44 (d, J = 2.1 Hz, 1H, H-5), 8.22 (dd, J = 8.9,2.1 Hz, 1H, H-7), 7.46 (d, J = 9.0 Hz, 1H, H-8), 4.39-4.31 (m, 2H, H-1a), 4.23 (q, J = 7.2 Hz, 2H, H-3c), 2.63 (s, 3H, H-6b), 1.50 (t, J = 7.3 Hz, 3H, H-3d), 1.37-1.20 (m, 3H, H-1b). ¹³C NMR (75 MHz, CDCl₃) d 196.9 (6a), 173.9 (4), 165.4 (3a), 149.1 (2), 141.6 (3), 133.3 (6), 131.4 (5), 129.5 (7), 128.7 (4a), 116.2 (8), 112.4 (8a), 61.1 (3c), 49.1 (1a), 26.6 (6b), 14.5 (1b), 14.4 (3d). IR (neat, cm^-1): 3053, 2982, 2921,1712,1685, ESI-HRMS m/z [M+H]+ calcd for C₁₆H₁₈NO₄ 288.1230, found 288.1234.

Methyl 6-acetyl-4-oxo-1-(4-(trifluoromethyl)benzyl)-1,4-dihydro-quinoline-3-carboxylate, 3e

Brown powder, 0.680 g (68 %), R = 0.81 (DCM/MeOH 10:1), m.p. 133-136 °C; ¹H NMR (300 MHz, DMSO) d 9.02 (s, 1H, H-2), 8.76 (d, J = 3.4 Hz, 1H, H-5) 8.15 (dd, J = 9.1,3.4 Hz, 1H, H-7), 7.73 (d, J = 8.0, Hz, 2H, H-1c), 7.60 (d, J = 7.1 Hz, 2H, H-1d), 7.43 (d, J = 9.1 Hz, 1H, H-8), 5.82 (s, 2H, H-1a), 3.89 (s, 3H, H-3c), 2.49 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 173.9 (4), 164.4 (3a), 151.4 (C-2), 142.2 (C-3), 140.2 (C-1b), 135.7 (C-1d), 133.3 (C-6), 132.3 (C-1c), 132.0 (C-5), 129.2 (C-7), 128.2 (C-1e), 127.6 (C-4a), 121.5 (C-8), 118.8 (C-8a), 111.6 (C-1f), 55.3 (C-3c), 53.7 (C-1a), 26.6 (6b). IR (neat, cm^-1): 3100, 2970, 2921, 1700, 1680. ESI-HRMS m/z [M+H]+ calcd for C₂₁H₁₆F₃NO₄ 404.1104, found 404.1107.

Ethyl 6-acetyl-1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate, 3f

White powder, 0.80 g (58 %), R = 0.81 (DCM/MeOH 10:1) m.p. 142-144 °C; ¹H NMR (300 MHz, DMSO) d 9.00 (s, 1H, H-2), 8.81 (s, 1H, H-5), 8.19 (d, J = 8.8 Hz, 1H, H-8), 7.77 (d, J = 8.9 Hz, 1H, H-7), 7.49-7.21 (m, 5H, H-1c/H-1g), 5.77 (s, 2H, H-1a), 4.31 (q, J= 7.0 Hz, 2H, H-3c), 2.55 (s, 3H, H-6b), 1.40-1.26 (m, 3H, H-3d). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 173.3 (C-4), 164.7 (C-3a), 151.1 (C-2), 142.4 (C-3), 136.2 (C-1b), 133.2 (C-6), 131.8 (C-5), 129.4 (C-1g), 128.4 (C-4a), 128.3 (C-1f), 127.7 (C-7), 126.9 (C-1e), 118.8 (C-8), 111.9 (C-8a), 60.5 (C-3c), 56.2 (C-1a), 27.2 (C-6a), 14.7 (C-3d). IR (neat, cm^-1): 3083, 2970, 2921, 1705, 1681, ESI-HRMS m/z [M+H]+ calcd for C₂₁H₂₀NO₄ 350.1387, found 350.1391.

Brown powder, 0.58 g (62 %), R = 0.81 (DCM/MeOH 10:1), m.p. 173-175 °C; ¹H NMR (300 MHz, DMSO) d 8.99 (s, 1H, H-2), 8.75 (s, 1H, H-5), 8.16 (d, J = 8.6 Hz, 1H, H-7), 7.81-7.52 (m, 3H, H-1c, H-8), 7.19 (d, J = 7.6 Hz, 2H, H-1d), 5.72 (s, 2H, H-1a), 3.79 (s, 3H, H-3c), 2.50 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.4 (C-6a), 173.1 (C-4), 166.4 (C-3a), 151.4 (C-2), 142.2 (C-3), 135.7 (C-1d), 133.3 (C-6), 132.3 (C-1c), 132.0 (C-5), 129.2 (C-7), 128.3 (C-1b), 127.6 (C-4a), 127.5 (C-8), 119.8 (C-8a), 111.6 (C-1e), 55.3 (C-3c), 53.7 (C-1a), 26.6 (6b). IR (neat, cm^-1): 3048,2972,2931,1702, 1682. ESI-HRMS m/z [M+H]+ calcd for C₂₀H₁₇BrNO₄ 414.0335, found 414.0333.

Methyl 6-acetyl-1-(2,4-dichlorobenzyl)-4-oxo-1,4-dihydroquino-line-3-carboxylate, 3h

Brown powder, 0.28 g (48 %), R = 0.81 (DCM/MeOH 10:1), m.p. 197-1199 °C; ¹H NMR (300 MHz, DMSO) d 9.16 (s, 1H, H-2), 8.92 (d, J = 2.2 Hz, 1H, H-5), 8.26 (dd, J = 8.9,2.2 Hz, 1H, H-7), 7.85 (d, J = 9.0 Hz, 1H, H-8), 7.72-7.59 (m, 2H, H-1d, H-1g), 7.21 (dd, J = 8.3,2.2 Hz, 1H, H-1f), 5.87 (s, 2H, H-1a), 3.77 (s, 3H, H-3c), 2.70 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.3 (C-4), 164.0 (C-3a), 150.2 (C-2), 142.1 (C-3), 137.4 (C-6), 133.4 (C-1b), 132.1 (C-5), 131.9 (C-1g), 131.6 (C-1c), 131.1 (C-1e), 129.4 (C-1d), 127.7 (C-7), 127.4 (C-1f), 127.2 (C-4a), 118.7 (C-8), 112.8 (C-8a), 55.5 (C-1a), 52.3 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3073, 2972, 2941, 1702, 1683. ESI-HRMS m/z [M + H]+ calcd for C₂₀H₁₆Cl₂NO₄ 404.0451, found 404.0452.

4.2.2. General Method for the Preparation of Amides 4a-j

A mixture of 3 (1 g, 1 eq.), DBU (320 //L, 0.33 g, 2.1 mmol), an appropriate amine (5 eq.), and chloroform (15 mL) in a 100 mL round-bottom flask was stirred under reflux for 24-30 h.³⁴ Upon reaction completion as indicated by TLC, the mixture was evaporated to dryness and resultant crude subjected to silica gel column chromatography eluting with CH₂Cl₂/MeOH (10:1). Fractions containing the desired product were combined, evaporated to dryness and recrystallized from ethanol. Compounds 4a-j were obtained in 30-50 % yield following this procedure.

6-Acetyl-1-ethyl-N-(2-methoxyethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, 4a

Orange powder, 0.028 g (32 %), R = 0.48 (DCM/MeOH 10:1), m.p. 163-165 °C; ¹H NMR (300 MHz, DMSO) d 9.93 (s, 1H, Nh), 8.88 (s, 1H, H-2), 8.83 (d, J = 1.5 Hz, 1H, H-5), 8.32-8.22 (m, 1H, H-7), 7.97 (d, J = 9.0 Hz, 1H, H-8), 4.55 (q, J = 7.0 Hz, 2H, H-1a), 3.49 (s, 3H, H-3f), 3.42-3.04 (m, 4H, H-3c, H-3d), 2.68 (s, 3H, H-6b), 1.39 (t, J = 7.0 Hz, 3H, H-1b). ¹³C NMR (75 MHz, DMSO) d 197.6 (C-6a), 175.9 (C-4), 164.2 (C-3a), 148.8 (C-2), 142.1 (C-3), 132.9 (C-6), 132.1 (C-5), 127.9 (C-7), 127.1 (C-4a), 118.4 (C-8), 112.6 (C-8a), 71.3 (C-3d), 58.3 (C-3f), 49.1 (C-1a), 38.8 (C-3c), 27.5 (C-6b), 14.9 (C-1b). IR (neat, cm^-1): 3393, 3041, 2970, 2929, 1682, 1656. ESI-HRMS m/z [M+H]+ calcd for C₁₇H₂₁N₂O₄ 317.1496, found 317.1497.

6-Acetyl-1-ethyl-N-(2-(2-hydroxyethoxy)ethyl)-4-oxo-1,4-dihydro-quinoline-3-carboxamide, 4b

White powder, 0.235 g (33 %), R = 0.39 (DCM/MeOH 10:1), m.p. 127-129 °C; ¹H NMR (400 MHz, CDCl₃) d 10.12 (s, 1H, NH), 8.97 (s, 1H, H-2), 8.75 (s, 1H, H-5), 8.27 (d, J = 7.9 Hz, 1H, H-7), 7.54 (d, J =7.9Hz,1H,H-8),4.31 (q, J =7.1Hz,2H,H-1a),3.74-3.61 (m, 8H, H-3c, H-3d, H-3f, H-3g), 2.67 (s, 3H, H-6b), 1.53 (t, J = 7.2 Hz, 3H, H-1b). ¹³C NMR (101 MHz, CDCl₃) d 196.8 (C-6a), 176.7 (C-4), 164.7 (C-3a), 147.9 (C-2), 141.5 (C-3), 133.6 (C-6), 131.9 (C-5), 129.2 (C-7), 127.9 (C-4a), 116.2 (C-8), 113.5 (C-8a), 72.7 (C-3g), 69.7 (C-3f), 61.7 (C-3d), 49.3 (C-1a), 39.0 (C-3c), 26.6 (C-6b), 14.6 (C-1b). IR (neat, cm^-1): 3333,3252,3001,2970,2929,1682,1654. ESI-HRMS m/z [M+H]+ calcd for C₁₈H₂₃N₂O₅ 347.1601, found 347.1604.

6-Acetyl-1-ethyl-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, 4c

White powder, 0.475 g (48 %), R = 0.11 (DCM/MeOH 10:1), m.p. 157-159 °C; ¹H NMR (300 MHz, DMSO) d 9.97 (t, J = 5.9 Hz, 1H, NH-3b), 8.91 (s, 1H, H-2), 8.84 (d, J = 2.2 Hz, 1H, H-5), 8.30 (dd, J = 9.0,2.2 Hz, 1H, H-7), 8.01 (d, J = 9.0 Hz, 1H, H-8), 5.27 (s, 1H, H-3h), 4.56 (q, J = 7.1 Hz, 2H, H-1a), 3.67 (t, J = 6.3 Hz, 4H, H-3d, H-3f), 3.15 (t, J = 8.8 Hz, 2H, H-3g), 3.03 (t, J = 5.4 Hz, 2H, H-3c), 2.68 (s, 3H, H-6b), 1.39 (t, J = 7.1 Hz, 3H, H-1b). ¹³C NMR (75 MHz, DMSO) d 197.2 (C-6a), 175.8 (C-4), 165.1 (C-3a), 149.2 (C-2), 141.8 (C-3), 133.2 (C-6), 132.2 (C-5), 127.6 (C-7), 127.2 (C-4a), 118.5 (C-8), 112.3 (C-8a), 56.9 (C-3g), 49.7 (C-1a), 49.0 (C-3c), 47.1 (C-3f), 35.9 (C-3d), 27.2 (C-6b), 14.9 (C-1b). IR (neat, cm^-1): 3313, 3243, 3081,2879,2819,1687,1656. ESI-HRMS m/z [M+H]+ calcd for C₁₈H₂₄N₃O₄ 346.1761, found 346.1762.

N-(3-(1H-imidazol-1-yl)propyl)-6-acetyl-1-ethyl-4-oxo-1,4-dihydro-quinoline-3-carboxamide, 4d

White powder, 0.521 g (57 %), R = 0.4 (DCM/MeOH 10:1), m.p. 207-209 °C; ¹H NMR (300 MHz, CDCl₃) d 9.98 (s, 1H, NH), 8.99 (s, 1H, H-2), 8.76 (d, J = 0.9 Hz, 1H, H-5), 8.28 (dd, J = 9.0,0.9 Hz, 1H, H-7), 7.57 (d, J = 9.0 Hz, 1H, H-8), 7.49 (s, 1H, H-3g), 6.99 (d, J = 9.2 Hz, 1H, H-3j), 6.93 (d, J = 9.2 Hz, 1H, H-3i), 4.32 (q, J = 7.2 Hz, 2H, H-1a), 4.01 (t, J = 7.0 Hz, 2H, H-3e), 3.43 (q, J = 6.3 Hz, 2H, H-3c), 2.66 (s, 3H, H-6b), 2.05 (dt, J = 6.8, 6.3 Hz, 2H, H-3d), 1.54-1.49 (t, J = 7.2 Hz, 3H, H-1b). ¹³C NMR (75 MHz, CDCl3) d 196.6 (C-6a), 176.5 (C-4), 164.8 (C-3a), 147.9 (C-2), 141.6 (C-3), 137.2 (C-3g), 133.4 (C-6), 131.7 (C-5), 129.5 (C-3j), 128.9 (C-7), 127.4 (C-4a), 118.9 (C-3i), 116.4 (C-8), 112.9 (C-8a), 49.4 (C-1a), 44.5 (C-3c), 36.1 (C-3e), 31.3 (C-3d), 26.6 (C-6b), 14.6 (C-1b). IR (neat, cm^-1): 3303, 3087, 2960, 2912, 1687, 1655. ESI-HRMS m/z [M+H]+ calcd for C₂₀H₂₃N₄O₃ 367.1765, found 367.1767.

6-Acetyl-1-(4-bromobenzyl)-N-(2-methoxyethyl)-4-oxo-1,4-dihydro-quinoline-3-carboxamide, 4e

White powder, 0.432 g (43 %), R = 0.4 (DCM/MeOH 10:1), m.p. 136-138 °C; ¹H NMR (300 MHz, DMSO) d 9.94 (t, J = 5.0 Hz, 1H, NH), 9.10 (s, 1H, H-2), 8.86 (d, J = 2.1 Hz, 1H, H-5), 8.19 (dd, J = 9.0, 2.2 Hz, 1H, H-7), 7.79 (d, J = 9.0 Hz, 1H, H-8), 7.62-7.45 (m, 2H, H-1d), 7.29-7.16 (m, 2H, H-1c), 5.79 (s, 2H, H-1a), 3.51 (t, J = 4.6 Hz, 2H, H-3c), 3.48-3.06 (m, 5H, H-3d, H-3f), 2.65 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.2 (C-4), 164.1 (C-3a), 150.2 (C-2), 142.2 (C-3), 135.7 (C-1b), 133.3 (C-6), 132.3 (C-5), 132.0 (C-1c), 129.2 (C-1d), 127.7 (C-7), 127.3 (C-1e), 121.5 (C-4a), 118.9 (C-8), 112.7 (C-8a), 71.2 (C-3d), 58.5 (C-3f), 55.8 (C-1a), 38.8 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3317, 3051, 2960, 2900,1686,1657. ESI-HRMS m/z [M+H]+ calcd for C₂₂H₂₂BrN₂O₄ 459.0763, found 459.0751.

6-Acetyl-1-(4-bromobenzyl)-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, 4f

White powder, 0.20 g (48 %), R = 0.11 (DCM/MeOH 10:1), m.p. 178-180 °C; ¹H NMR (300 MHz, DMSO) d 9.93 (t, J = 4.8 Hz, 1H, NH), 9.10 (s, 1H, H-2), 8.85 (d, J = 1.7 Hz, 1H, H-5), 8.19 (dd, J = 8.9,1.7 Hz, 1H, H-7), 7.79 (d, J = 9.0 Hz, 1H, H-8), 7.54 (d, J = 8.3 Hz, 2H, H-1d), 7.20 (d, J = 8.3 Hz, 2H, H-1c), 5.79 (s, 2H, H-1a), 4.62 (t, J = 5.2 Hz, 2H, H-3c), 3.60-3.49 (m, 6H, H-3d, H-3f, H-3g), 2.43 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.2 (C-4), 164.1 (C-3a), 150.2 (C-2), 142.2 (C-3), 135.7 (C-1d), 133.4 (C-6), 132.2 (C-1c), 132.0 (C-5), 129.3 (C-7), 127.4 (C-1b), 121.6 (C-4a), 118.9 (C-8), 112.7 (C-8a), 111.9 (C-1e), 58.7 (C-3g), 55.8 (C-1a), 49.4 (C-3f), 48.9 (C-3d), 36.9 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3320, 3202, 3000, 2970, 2929, 1682, 1657. ESI-HRMS m/z [M+H]+ calcd for C₂₃H₂₅BrN₃O₄ 486.1023, found 486.1021.

6-Acetyl-1-(4-bromobenzyl)-N-[2-(2-hydroxyethoxy)ethyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide, 4g

White powder, 0.20 g (48 %), R = 0.4 (DCM/MeOH 10:1), m.p. 183-185 °C; ¹H NMR (300 MHz, DMSO) d 9.93 (t, J = 4.8 Hz, 1H, NH), 9.10 (s, 1H, H-2), 8.85 (d, J = 1.7 Hz, 1H, H-5), 8.19 (dd, J = 8.9,1.7 Hz, 1H, H-7), 7.79 (d, J = 9.0 Hz, 1H, H-8), 7.54 (d, J = 8.3 Hz, 2H, H-1d), 7.20 (d, J = 8.3 Hz, 2H, H-1c), 5.79 (s, 2H, H-1a), 4.62 (t, J = 5.2 Hz, 2H, H-3c), 3.60-3.49 (m, 6H, H-3d, H-3f, H-3g), 2.43 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.2 (C-4), 1- 64.1 (C-3a), 150.2 (C-2), 142.2 (C-3), 135.7 (C-1d), 133.4 (C-6), 132.2 (C-1c), 132.0 (C-5), 129.3 (C-7), 127.4 (C-1b), 121.6 (C-4a), 118.9 (C-8), 112.7 (C-8a), 111.9 (C-1e), 72.7 (C-3d), 69.7 (C-3f), 60.7 (C-3g), 55.0 (C-1a), 39.7 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3397, 3252, 3041, 2950, 2861, 1686, 1654. ESI-HRMS m/z calcd for C₂₃H₂₄BrN₂O₅ 487.0863, found 487.0665 [M+H]+. HPLC purity > 96 %, retention time = 9.89 min.

6-Acetyl-1-benzyl-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, 4h

Orange powder, 0.320 g (48 %), R = 0.11 (DCM/MeOH 10:1), m.p. 169-171 °C; ¹H NMR (300 MHz, DMSO) d 9.93 (t, J = 4.8 Hz, 1H, NH), 9.10 (s, 1H, H-2), 8.85 (d, J =1.7 Hz, 1H, H-5), 8.19 (dd, J = 8.9,1.7 Hz, 1H, H-7), 7.79 (d, J = 9.0 Hz, 1H, H-8), 7.54-7.20 (m, 5H, H-1c, H-1d, H-1f, H-1g), 5.79 (s, 2H, H-1a), 4.62 (t, J = 5.2 Hz, 2H, H-3c), 3.60-3.49 (m, 6H, H-3d, H-3f, H-3g), 2.43 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.2 (C-4), 164.1 (C-3a), 150.2 (C-2), 142.2 (C-3), 135.7 (C-1d), 133.4 (C-6), 132.2 (C-1c), 132.0 (C-5), 129.3 (C-7), 127.4 (C-1b), 121.6 (C-4a), 118.9 (C-8), 112.7 (C-8a), 111.9 (C-1e), 72.7 (C-3d), 69.7 (C-3f), 60.7 (C-3g), 55.02 (C-1a), 39.7 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3328, 3212, 3038,2971,2921,1682,1659. ESI-HRMS m/z [M+H]+ calcd for C₂₃H₂₆N₃O₄ 408.1918, found 408.1923.

6-Acetyl-1-(2,4-dichlorobenzyl)-N-(2-methoxyethyl)-4-oxo-1,4-dihy-droquinoline-3-carboxamide, 4i

Brown powder, 0.370 g (44 %), R = 0.4 (DCM/MeOH 10:1). m.p. 203-205 °C; ¹H NMR (300 MHz, DMSO-d₆) d 9.99 (t, J = 5.1 Hz, 1H, NH), 9.16 (s, 1H, H-2), 8.92 (d, J = 2.2 Hz, 1H, H-5), 8.26 (dd, J = 8.9, 2.2 Hz, 1H, H-7), 7.85 (d, J = 9.0 Hz, 1H, H-8), 7.72-7.59 (m, 2H, H-1d, H-1f), 7.21 (d, J = 8.3, Hz, 1H, H-1g), 5.87 (s, 2H, H-1a), 3.56-3.13 (m, 4H, H-3c, H-3d), 3.37 (s, 3H, H-3f), 2.70 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.3 (C-4), 164.0 (C-3a), 150.2 (C-2), 142.1 (C-3), 137.4 (C-6), 133.4 (C-1b), 132.1 (C-5), 131.9 (C-1g), 131.6 (C-1c), 131.1 (C-1e), 129.4 (C-1d), 127.7 (C-7), 127.4 (C-1f), 127.2 (C-4a), 118.7 (C-8), 112.8 (C-8a), 71.2 (C-3d), 58.5 (C-3f), 55.3 (C-1a), 39.0 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3298,3071,2971,2929,1682,1655. ESI-HRMS m/z [M+H]+ calcd for C₂₂H₂₁Cl₂N₂O₄ 447.0873, found 447.0878.

6-Acetyl-N-(2-((2-hydroxyethyl)amino)ethyl)-4-oxo-1-(4-(trifluoro-methyl)benzyl)-1,4-dihydroquinoline-3-carboxamide, 4j

Orange powder, 0.367 g (44 %), R = 0.11 (DCM/MeOH 10:1), m.p. 193-195 °C; ¹H NMR (300 MHz, DMSO) d 10.03 (t, J = 5.9 Hz, 1H, NH), 9.22 (s, 1H, H-2), 8.92 (d, J = 2.1 Hz, 1H, H-5), 8.28 (dd, J = 8.9, 2.2 Hz, 1H, H-7), 7.87 (d, J = 9.0 Hz, 1H, H-8), 7.79 (d, J = 8.1 Hz, 2H, H-1c), 7.51 (d, J = 8.0 Hz, 2H, H-1d), 6.03 (s, 2H, H-1a), 5.36 (t, J = 5.1 Hz, 1H, OH), 3.83-3.70 (m, 4H, H-3d, H-3f), 3.22 (t, J = 6.3 Hz, 2H, H-3c), 3.11 (t, J = 5.3 Hz, 2H, H-3g), 2.70 (s, 3H, H-6b). ¹³C NMR (75 MHz, DMSO) d 197.1 (C-6a), 176.1 (C-4), 165.0 (C-3a), 150.4 (C-2), 142.2 (C-3), 141.1 (C-1b), 133.5 (C-6), 132.4 (C-5), 129.1 (C-1e), 127.7 (C-7), 127.4 (C-1c), 127.3 (C-1d), 126.3 (C-1f), 126.3 (C-4a), 118.9 (C-8), 112.6 (C-8a), 56.9 (C-3g), 55.9 (C-1a), 49.6 (C-3f), 47.0 (C-3d), 35.9 (C-3c), 27.2 (C-6b). IR (neat, cm^-1): 3293, 3252, 3081, 2974, 2926, 1682, 1654. ESI-HRMS m/z [M+H]+ calcd for C₂₄H₂₅F₃N₃O₄ 476.1792, found 476.1797.

4.3. In vitro Anti-trypanosomal Assay

Trypanosoma brucei brucei 427 trypomastigotes were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Lonza) supplemented with 10 % fetal calf serum,³⁵ HMI-9 supplement, hypoxanthine and penicillin/streptomycin at 37 °C ina5%CO₂ incubator. Serial dilutions of test compounds were incubated with the parasites in 96-well plates for 24 h and residual parasite viability in the wells determined by adding 20 of 0.54 mM resazurin in phosphate buffered saline (PBS) and incubating for an additional 2-4 h. Reduction of resazurin to resorufin by viable parasites was assessed by fluorescence readings (excitation 560 nm, emission 590 nm) in a Spectramax M3 plate reader. Fluorescence readings were converted to % parasite viability relative to the average readings obtained from untreated control wells. IC₅₀ values were determined by plotting % viability vs. log[compound] and performing non-linear regression using GraphPad Prism (v. 5.02) software.

4.4. In vitro Cytotoxicity Assay

HeLa cells (Cellonex) were cultured in Dulbecco's modified Eagle's medium (DMEM; Lonza) supplemented with 10 % fetal calf serum and antibiotics (penicillin/streptomycin/ampho-tericin B) at 37 °C ina5%CO₂ incubator. Cells were plated in 96-well plates at a cell density of2x10⁴ cells per well and grown overnight. Serial dilutions of test compounds were incubated with the cells for an additional 24 h, and cell viability in the wells assessed by adding 20 0.54 mM resazurin in PBS for an additional 2-4 h. Fluorescence readings (excitation 560 nm, emission 590 nm) obtained for the individual wells were converted to % cell viability relative to the average readings obtained from untreated control wells. Plots of % cell viability vs. log[com-pound] were used to determine IC₅₀ values by non-linear regression using GraphPad Prism (v. 5.02).

 

Supplementary Material

Supplementary information is provided in the online supplement.

 

Acknowledgements

The authors acknowledge the financial support by the National Research Foundation (SDK), Rhodes University for a Postdoctoral fellowship (RMB) and Rhodes University Sandisa Imbewu (SDK) towards this research. The bioassay component of the project was funded by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package awarded to HCH.

ORCID iDs

S.D. Khanye: © orcid.org/0000-0003-0725-5738 R.M. Beteck: © orcid.org/0000-0002-6282-043X

 

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Received 7 March 2018
Revised 13 November 2018
Accepted 15 November 2018

 

 

* To whom correspondence should be addressed. E-mail: r.beteck@ru.ac.za/s.khanye@ru.ac.za

 

 

Supplementary Data

The supplementary data is available in pdf: [Supplementary data]