https://orcid.org/0000-0001-6455-5716
Abstract
Carbonic anhydrases (CAs) are one of the promising targets for the development of anticancer agents. CA isoforms are implicated in various physiological processes and are expressed in both normal and cancerous cells. Thus, non-isoform selective inhibitors are associated with several side effects. Consequently, designing selective inhibitors towards cancer-related hCA IX/XII rather than the ubiquitous cytosolic isozymes hCA I and II is the main research objective in the field. Herein, a new series of 3-(6-methylpyridin-2-yl)coumarin derivatives 3 and 5a–o was designed and synthesised. The CA inhibition activities for the synthesised coumarins were analysed on isoforms hCA I, II, IX, and XII. Interestingly, both cancer-linked isoforms hCA IX/XII were inhibited by the prepared coumarins with inhibition constants ranging from sub- to low-micromolar range, whereas hCA I and II isoforms haven’t been inhibited up to 100 µM. Furthermore, the target coumarins were assessed for their antitumor activity on NCI-59 human cancer types.
1. Introduction
Carbonic anhydrases (CAs) are vital for the processes of CO2 hydration and HCO3- dehydrationCitation1–3. The α-CAs are one of the seven known CAs families which are predominantly found in vertebrates, green plants cytoplasm, bacteria, and algaeCitation4,Citation5. Among the sixteen human carbonic anhydrases (hCAs) isozymes found, the hCA IX and XII play a crucial role in the cancer cell persistence by controlling the intracellular pH; thus, their inhibitors are deemed to be an efficient antitumor approachCitation4,Citation6. hCA IX expression is associated with a bad prognosis in cancer, whereas hCA XII isozyme is expressed in normal tissues and overexpressed in a variety of malignanciesCitation7–10. Furthermore, non-selective inhibition of hCAs leads to some side effects while treating cancerCitation11. Consequently, designing selective inhibitors of hCA IX/XII rather than the ubiquitous cytosolic isozymes hCA I and II is the main target.
Classical CA inhibitors (CAIs) are mostly based on a sulphonamide moiety as a zinc-binding group (ZBG) among which the clinically used CAIs; such as acetazolamide and methazolamide. On the other hand, the non-classical CAIs do not rely on ZBGCitation11,Citation12. Among the non-classical CAIs; coumarins, carboxylic acids, phenols, and polyamines can inhibit the catalytic activity of CA by different mechanisms rather than coordinating to the zincCitation13,Citation14.
Coumarin ring, as a privileged scaffold, exerted exceptional anticancer profile acting through various mechanisms of actionCitation15,Citation16. Coumarin (I, ) derivatives were introduced by Supuran’ group as a non-classical type of CAIsCitation17. Coumarin was shown to undergo hydrolysis to form cis-2-hydroxy-cinnamic acid (II, ), instead of binding the CA active site with its intact coumarin moiety. The substantial selective inhibitory effect towards hCA IX and XII is attributable to the binding of the hydrolysis product II to the amino acid residues constituting the rim of the active site cavity, which differed significantly between different hCA isoformsCitation12,Citation17,Citation18. These findings grasped the attention for developing a variety of coumarin-based CAIs, such as compounds III–V (), which exerted efficient and selective inhibition activity towards the cancer-related isozymes IX and XII over the constitutional isozymes CA I and IICitation18–20.
Figure 1. Structure of coumarin I, its hydrolysed form II, some reported coumarin-based CAIs III–V, some reported pyridine derivatives bearing chalcone functionality VI–IX, and target compounds 5a–o.
On the other hand, pyridine ring is identified as a valuable scaffold for the development of a wide range of approved drugs especially the anticancer ones such imatinibCitation21, sorafenibCitation22, and acalabrutinibCitation23. The pyridine-based small molecules bearing chalcone functionality VI-VIII () have been described for their in vitro anticancer activity against different cancer cell linesCitation24–27. In addition, the pyridine derivatives VIII and IX were able to inhibit the cancer-related CA IX isoform selectivelyCitation24–27.
In this work, the design and synthesis of a series of small molecules based on 3-(6-methylpyridin-2-yl)-coumarin (MPC) scaffold as potential selective cancer-associated CA isoform IX/XII inhibitors was achieved (). The design of target MPCs relies on the incorporation of the coumarin moiety which can exert the CA inhibitory action through obstructing the entry of the active site cavity. Thereafter, the acetyl-bearing pyridine motif was embedded on the coumarin ring as a privileged scaffold in cancer drug discovery to provide MPC ketone 3, which utilised to prepare the target MPC chalcones (5a–o, ). The newly prepared series included different lipophilic aromatic rings spanning various ring sizes and different substituents on the aromatic ring, that anticipated to afford lipophilic interactions with the amino acid residing of the rim of the CA active site. The herein synthesised target MPCs were evaluated for their CA inhibition activity as well as for their antiproliferative activity towards different 59 cancer cell lines in the US-NCI.
2. Results
2.1. Chemistry
The synthesis strategy for MPC 3 and 5a–o construction is illustrated in Schemes 1 and 2. 3-Acetylcoumarin 1 was prepared by Knoevenagel condensation through the reaction of salicylaldehyde with ethyl acetoacetate in the presence of piperidine (few drops) as a catalyst according to the reported methodCitation28. The reaction of 3-acetylcoumarin 1 with dimethyl formamide dimethyl acetal (DMF-DMA) under reflux temperature in dry toluene gave the strategic starting material enaminone 2.
Scheme 1. Reagents and conditions: (i) Dry toluene, reflux 7 h.; (ii) Acetylacetone, CH3COONH4, AcOH, reflux 10 h.
The condensation of 2 with acetylacetone and ammonium acetate in refluxing acetic acid yielded 3-(5-acetyl-6-methylpyridin-2-yl)-2H-chromen-2-one 3. The chalcones 5a–o can be readily synthesised via the classical base-catalyzed Claisen–Schmidt condensation reaction through the reaction of ketone 3 with various aromatic aldehydes 4a–o in a mixture of dioxane and methanol as a solvent at 0 °C (Scheme 2).
Scheme 2. Reagents and conditions: (i) KOH (aq.), dioxane: MeOH stirring at 0 °C 2 h then r.t overnight.
Sixteen compounds were synthesised in this study, and their structures were confirmed by using IR, 1H NMR, and 13C NMR (see the Supplementary Material). The elemental analysis results coincide with the molecular formula of target compounds within the accepted range (±0.04%). In the predicted regions of NMR spectra, the methyl (–CH3), methylene (–CH2–), and methoxy (–OCH3) group signals appeared in the aliphatic region for both protons and carbons spectra of the corresponding targets.
2.2. Biological evaluation
2.2.1. Carbonic anhydrase isoforms inhibition assay
The newly synthesised MPCs (3 and 5a–o) were assessed for their CA inhibition activity employing the stopped-flow CO2 hydrase assayCitation29 for constitutional hCA (I/II) isoforms and cancer-linked hCA (IX/XII) isoforms. Inhibition values given in revealed that the herein-reported MPCs have varying degrees of inhibitory action against the examined CA isoforms.
Table 1. Inhibition data of MPC 3 and 5a–o against hCA isoforms I, II, IX, and XII using AAZ as a reference. 
The examined MPCs displayed one-digit micromolar inhibitory activity against the target cancer-linked isoform IX (KIs: 0.95–8.5 µM), except coumarins 5e, 5g, 5h, 5i, 5k, and 5o which displayed two-digit micromolar inhibition activity (KIs: 10.7–36.9 µM). It is worth noting that the acetyl derivative MPC 3 showed the most potent inhibitory action among the tested MPCs with sub-micromolar KI of 0.95 µM. MPC 5a endowed with an unsubstituted phenyl ring displayed low micromolar inhibitory activity (KI = 1.5 µM). In addition, the bioisosteric replacement of the phenyl moiety in 5a with different hetero moieties, such as pyridin-2-yl (5l), thiophen-2-yl (5m), and 5-methylfuran-2-yl (5n) maintained the low micromolar activity towards hCA IX isoform (KIs = 3.8, 1.1, and 1.5 µM, respectively). On the other hand, replacement of the phenyl ring with fused moieties, such as 1,3-benzodioxol-5-yl and naphtha-1-yl, led to about 3.5- and 23-fold decreased inhibitory activity (compounds 5j and 5k; KIs = 5.3 and 36.9 µM, respectively).
Moreover, the inhibition potency against hCA IX was found to be decreased with varying the size of substituents on the appended phenyl ring in the order of F > CH3 > Cl > N(CH3)2 > OCH3 > NO2, highlighting that incorporation of small substituents is further valuable for hCA IX inhibitory activity over the bulkier ones. In this context, grafting a morpholino or tri-methoxy substituents resulted in the decrease of the activity (compounds 5g and 5i; KIs = 12.0 and 27.4 µM, respectively) in comparison to the unsubstituted phenyl-bearing analogue 5a (KIs = 1.5 µM).
Further analysis of the inhibition data against hCA XII () revealed that the target MPCs 5a–o were able to affect this isoform with inhibition constants ranging from to sub-micromolar to low micromolar (KIs: 0.92–8.2 µM), except MPCs 5e, 5i, 5k, and 5o which displayed higher inhibition constant values (KIs = 10.9, 12.9, 21.4, and 17.8 µM, respectively). Among the examined MPC chalcones 5a–o, compound 5l emerged as the unique sub-micromolar hCA XII inhibitor (KI = 0.92 µM). In addition, MPCs 5c, 5g, and 5n showed potent inhibitory action with low inhibition constants equal 1.9, 1.8, and 1.9 µM, respectively.
It is worth mentioning that incorporation of an unsubstituted phenyl moiety led to MPC 5a with moderate hCA XII inhibitory action (KI = 5.1 µM), whereas grafting a halogen like para-fluoro (MPC 5b) and para-chloro (MPC 5c) improved the inhibitory activity (KIs = 2.7 and 1.9 µM, respectively) which highlights that halogens incorporation is beneficial for the hCA XII inhibitory effect. Moreover, grafting a para-morpholino or para-methoxy substituent elicited an enhanced activity (MPCs 5g and 5h; KIs = 1.8 and 2.8 µM, respectively) in comparison to the unsubstituted phenyl-bearing counterpart MPC 5a (KI = 5.1 µM). In addition, the bioisosteric replacement of phenyl motif in MPC 5a with different heterocycles, such as the pyridine (MPC 5l), thiophene (MPC 5m), and furan (MPC 5n) moieties boosted the hCA XII inhibitory action of the target MPC chalcones (KIs = 0.92, 2.7, and 1.9 µM, respectively). On the other hand, replacement of the phenyl moiety with the fused naphthyl carbocycle (MPC 5k; KI = 21.4 µM) or the bulky 3-methyl-1-phenyl-pyrazole heterocycle (MPC 5o; KI = 17.8 µM) exerted a worsening impact towards the hCA XII inhibitory activity.
It is worth stressing that MPC ketone 3 established the best inhibitory activity against both hCA IX and hCA XII isoforms in this study (KIs = 0.95 and 0.68 µM, respectively), hinting out the grafting small functionalities within the pyridine ring is more appropriate for the hCA inhibitory activity, and should be considered for further optimisation of MPC scaffold in the future research.
As expected, both hCA I and II isoforms were not inhibited by all newly synthesised MPCs which demonstrated inhibition constants more than 100 µM. Accordingly, all the designed MPCs showed excellent selectivity towards both cancer-related isoform IX and XII, compared with the cytosolic isoforms (). Selectivity index (SI) offered obviously presented that MPC ketone 3 showed the highest selectivity profile towards hCA IX over hCA I and II (SI > 105.26) and hCA XII over hCA I and II (SI > 147.06) followed by MPC chalcones 5m, 5n, and 5a, whereas the least selectivity was obtained by the bulky substituted derivatives 5i, 5k, and 5o.
Table 2. Selectivity ratios for MPC 3 and MPCs 5a–o towards cancer-related hCA isoforms.
2.2.2. NCI cancer cell lines screening
Following NCI protocol, sixteen MPCs were screened for their potential in vitro anticancer effects against human 59 cancer cell panels including prostate, leukaemia melanoma, colon, breast, CNS, renal, NSCLC, and ovarian cancers by National Cancer Institute (USA)Citation30.
2.2.3. Preliminary single (10 µM) dose screening
The antiproliferative activities of MPC 3 and MPCs 5a–o were first evaluated in a 10 µM dose assay, with SRB assay used to determine cell survival and proliferation. According to the SRB assay outcomes, most of the newly prepared MPCs exerted weak or non-significant anticancer activity towards the majority of examined cells have mean percentages growth inhibition (GI%) range 0–10%, except MPCs 5g and 5l which demonstrated good anti-proliferative activities towards different cancer cell lines (mean% GI = 28 and 50%, respectively). The results of the cell growth inhibitory activities for MPCs 5g and 5l towards the different treated tumour cell lines were presented as GI% and presented in .
Table 3. Cell growth inhibition (GI%) of 59 human tumour cell lines in vitro at a dose of 10 µM for MPCs 5g and 5l.
Assessing the obtained GI % values () revealed that MPC 5l is the most effective anti-proliferative agent among the compounds described here. The NCI screening results revealed anti-proliferative efficacy against 42 human cancer cell lines, indicating that this compound has broad-spectrum activity.
MPC 5l showed remarkable growth inhibition properties against Leukaemia (K-562/CCRF-CEM), Colon (HT29, KM 12, and SW-620), CNS (U251 and SF-539), Ovarian (IGROVI), Breast (MDA-MB-231 and MCF7) Renal (786-0) cancer cell lines, with inhibition % 93, 92, 93, 91, and 87%, respectively (). MPC 5l also showed strong efficacy towards leukaemia [MOLT-4/HL-60(TB)] and Renal (RXF 393) tumour cell lines, with inhibition percentages of 67, 72, and 67%, respectively. It is noteworthy that MPC 5l was shown to be lethal towards Leukaemia (RPMI-8226 and SR), Colon (HCT-15/HCT-116), and LOX IMVI Melanoma cells (GI % = 136, 112, 121, 133, and 184, respectively).
NCI screening results for MPC 5g showed anti-proliferative activity against 31 human cancer cell lines indicating a broad-spectrum activity. Compound 5g exerted its lethal action towards Melanoma MDAMB-435 cells with GI % = 121. Moreover, compound 5g exerted good activity towards Leukaemia [K-562, HL-60(TB), and SR] and (LOX IMVI) Melanoma cells (inhibition % 61, 62, 73, and 68, respectively). Additionally, compound 5g exerted moderate activity towards Colon cancer (HCT-15), CNS cancer (SF-539 and SNB-75), Melanoma (MALME-3M, M14 and UACC-62) and Breast (MDA-MB-468, MCF7, HS 578T, and MDA-MB-231) cancer cells with inhibition % 45, 54, 41, 42, 54, 47, 48, 45, 40, and 54, respectively ().
On the other hand, the obtained results for the remaining MPC chalcones 5a–f, 5h–k, and 5m–o ascribed to these derivatives selective actions towards certain cancer cell lines, as displayed in . In particular, compound 5b showed selective anticancer activity towards CNS cancer (SNB-75), Breast (MCF7), Melanoma (LOX IMVI) cells with inhibition % 39, 49, and 46, respectively. Also, compound 5f displayed good selectivity towards Melanoma (MDA-MB-435) cells (inhibition % = 80), whereas, compound 5n has selectivity towards Breast (MCF7) and Melanoma (LOX IMVI) cells (inhibition % 40 and 39, respectively).
Figure 2. The best anti-proliferative activities exerted by target MPC chalcones 5a–f, 5h–k, and 5m–o.
2.2.4. In vitro full NCI panel five dose assay
The preliminary single-dose assay results show that MPC 5l (NSC: 831974/1) is the most effective anticancer drug in this investigation, with promising inhibitory activity against a variety of cancer cell lines from various subpanels (). MPC 5l was then chosen for additional biological evaluation in a five-dose (0.01–100 µM) experiment. MPC 5l's GI50, TGI, and LC50 response parameters were obtained for each of the cancer cell lines studied. TGI represents cytostatic impact, whereas GI50 values reflect the extent of growth inhibitory effect. Furthermore, the LC50 parameter is regarded as the cytotoxicity parameter for the hybrid under investigation.
As shown in , MPC 5l had a potent anti-proliferative effect against nine human cancer cell lines tested: leukaemia (K-562, RPMI-8226, and SR), NSCLC (HOP-92), breast cancer (MCF7), colon (SW-620 and HCT-116) cancer, melanoma (LOX IMVI), and CNS (U251) with GI50 values ranging from 3.20 to 8.49 µM. MPC 5l, on the other hand, had GI50 > 100 µM against remaining cancer cells. Furthermore, MPC 5l demonstrated no cytostatic effect on all cancer cell lines (TGI > 100 µM). MPC 5l was discovered to be a non-lethal molecule with LC50 > 100 µM against all cancer cells.
Table 4. Results of the five-dose anticancer assay for MPC 5l against all fifty-nine cancer cell lines.
3. Conclusions
In brief, the present study demonstrates the design and synthesis of novel 6-(methylpyridin-2-yl)-coumarins MPC 3 and MPC (5a–o) as selective hCAIs. The synthesised target compounds selectively inhibited the cancer-related hCA isoforms with KI ranges: 0.95–36.9 µM (hCA IX) and 0.68–21.4 µM (hCA XII). All the designed MPCs showed excellent selectivity for hCA IX/hCA XII, over the cytosolic ones hCA I and hCA II with MPC 3 being the highest (SI towards hCA IX over hCA I and II > 105.26 and SI towards hCA XII over hCA I and II > 147.06). The SAR results emphasised that e grafting small functionalities within the pyridine ring is more appropriate for the hCA inhibitory activity. In vitro antitumor effects vs. various human cancer cells were also investigated, and 5l was found to have outstanding growth suppression characteristics against CNS, Colon, Ovarian, Breast, Leukaemia, and Renal cancer. MPC 5l was then chosen for further biological testing using a five-dose assay. The results showed that a single-digit micromolar concentration of the compound 5l had a potent anti-proliferative effect against nine human cancer cell lines, including leukaemia, NSCL cancer, colon cancer, CNS cancer, melanoma, and breast cancer, with GI50 values ranging from 3.20 to 8.49 µM.
4. Materials and methods
4.1. Chemistry
Melting points were measured in open-glass capillaries using a Stuart SMP30 apparatus at Tanta University's Faculty of Pharmacy's Central Research Laboratory in Tanta, Egypt. All organic chemicals and solvents were acquired from Sigma–Aldrich, Alfa Aesar, and Merck, respectively, and utilised without further purification. Analytical thin-layer chromatography (TLC): pre-coated aluminium sheets, 0.2 mm silica gel (Supelco Co., Silica 60 F254) used regularly to monitor reaction progress and ensure product purity utilising a developing system: The eluent was chloroform: methanol (2:1), which was visualised using a UV lamp set to 254 nm. The FT-IR spectra were detected on a ThermoFisher Scientific Nicolet-iS10 Spectrometer (MA, USA). 1H and 13C NMR spectra were carried out utilising the Bruker instrument at 400–500 MHz for 1H NMR and at 100–125 MHz for 13C NMR spectrophotometer, TMS is being used as an internal standard and chemical shifts were recorded in ppm on the δ scale using CDCl3-d as a solvent. The values of the coupling constant (J) were calculated in Hertz (Hz). The following are the split patterns: s, singlet; d, doublet; t, triplet; q, quartette; m, multiplet. Microanalysis was performed for C, H, and N elements on PerkinElmer 2400 (The regional centre for mycology and biotechnology, Al-Azhar University, Nasr City, Cairo, Egypt).
4.1.1. Synthesis of 3-[(2E)-3-(dimethylamino)prop-2-enoyl]-2H-chromen-2-one (2)
3-Acetyl-2-H-chromen-2-one 1 (1.88 g, 0.01 mol) and dimethylformamide-dimethylacetal (DMF-DMA) (1.19 g, 0.01 mol) were heated in dry toluene (10 ml) for 7 h at 110 °C. The cooled reaction mixture was filtered, washed with diethyl ether, dried, and crystallised from ethanol to yield compound 2 as a yellow powder (1.78 g, 73%). Mp: 159–161 °CCitation31.
4.1.2. Synthesis of 3-(5-acetyl-6-methylpyridin-2-yl)-2H-chromen-2-one (3)
In gl. AcOH (20 ml), an equimolar amount of enaminone 2 (1.70 g, 7 mmol), and acetylacetone (0.7 g, 7 mmol) was heated under reflux for 10 h in the presence of ammonium acetate (0.77 g, 10 mmol). The resultant product was collected, washed twice with water (2 × 10 ml), and recrystallized from acetonitrile to produce MPC ketone 3Citation32.
A yellow powder, yield: 70%. Mp: 208–210 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.63 (s, 3H, CH3), 2.84 (s, 3H, CO CH3), 7.34 (t, 1H, Arm. H, J = 8.0 Hz), 7.40 (d, 1H, Arm. H, J = 8.0 Hz), 7.59 (t, 1H, Arm. H, J = 8.0 Hz), 7.70 (d, 1H, Arm. H, J = 8.0 Hz), 8.08 (d, 1H, Arm. H, J = 8.0 Hz), 8.42 (d, 1H, Arm. H, J = 8.0 Hz), 8.93 (s, 1H, 4-H of coumarin ring).
4.1.3. General procedure for preparation of MPCs 5a–o
At 0 °C, a stirred solution of ketone 3 (0.5 mmol) and the suitable aldehyde (0.5 mmol) in a mixture of dioxane: methanol (4:2) (25 ml) was added to aqueous potassium hydroxide solution (0.15 g, in 1.5 ml dist. water).
The resulting mixture was agitated for 2 h at 0 °C before being warmed to room temperature overnight. The solvent was extracted under vacuum after the reaction was neutralised with gl. AcOH. MPCs 5a–o were produced by filtering the precipitate, washing it with diethyl ether, drying it, and crystallising it from ethanol.
4.1.3.1. 3-(6-Methyl-5-[(2E)-3-phenylprop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5a)
A yellow powder, yield: 85%. Mp: 207–209 °C. IR (νmax/cm−1): 3058 (CH-arom.), 2924, 2854 (CH-aliph.), 1724, 1665 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.74 (s, 3H, CH3), 7.16 (d, 1H, COCH=CH, J = 16.0 Hz), 7.35 (t, 1H, Arm. H, J = 8.0 Hz), 7.40–7.43 (m, 4H, Arm. H), 7.52 (d, 1H, COCH=CH, J = 16.0 Hz), 7.58–7.61 (m, 3H, Arm. H), 7.71 (d, 1H, Arm. H, J = 8.0 Hz), 7.90 (d, 1H, Arm. H, J = 8.0 Hz), 8.40 (d, 1H, Arm. H, J = 8.0 Hz), 8.91 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.77, 116.41, 119.42, 120.66, 124.50, 124.70, 125.98, 128.58 (2 C), 129.07 (2 C), 129.25, 131.08, 132.48, 133.55, 134.17, 136.57, 143.35, 146.88, 151.75, 153.98, 156.75, 160.40, 194.77. Anal. calcd. for C24H17NO3: C, 78.46; H, 4.66; N, 3.81. Found: C, 78.22; H, 4.61; N, 3.80.
4.1.3.2. 3-(5-[(2E)-3-(4-Fluorophenyl)prop-2-enoyl]-6-methylpyridin-2-yl)-2H-chromen-2-one (5b)
A pale-yellow powder, yield: 66%. Mp: 205–207 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.74 (s, 3H, CH3), 7.09 (d, 1H, COCH=CH, J = 16.0 Hz), 7.13 (d, 2H, Arm. H, J = 8.0 Hz), 7.36 (t, 1H, Arm. H, J = 8.0 Hz), 7.40 (d, 1H, Arm. H, J = 8.0 Hz), 7.49 (d, 1H, COCH = CH, J = 16.0 Hz), 7.57–7.61 (m, 3H, Arm. H), 7.71 (d, 1H, Arm. H, J = 8.0 Hz), 7.89 (d, 1H, Arm. H, J = 8.0 Hz), 8.40 (d, 1H, Arm. H, J = 8.0 Hz), 8.91 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.78, 116.23, 116.40, 116.43, 119.43, 120.67, 124.57, 124.72, 125.69, 129.10, 130.51, 130.57, 132.51, 133.45, 136.54, 143.38, 145.42, 151.81, 154.01, 156.80, 160.27, 163.33, 165.34, 194.44. Anal. calcd for C24H16FNO3: C, 74.80; H, 4.18; N, 3.63. Found: C, 74.97; H, 4.14; N, 3.59.
4.1.3.3. 3-(5-[(2E)-3-(4-Chlorophenyl)prop-2-enoyl]-6-methylpyridin-2-yl)-2H-chromen-2-one (5c)
A yellow powder, yield: 75%. Mp: 223–225 °C. IR (νmax/cm−1): 3064 (CH-arom.), 2966, 2925 (CH-aliph.), 1712, 1660 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.74 (s, 3H, CH3), 7.13 (d, 1H, COCH=CH, J = 16.0 Hz), 7.35 (t, 1H, Arm. H, J = 8.0 Hz), 7.39–7.41 (m, 3H, Arm. H), 7.46 (d, 1H, COCH = CH, J = 16.0 Hz), 7.51 (d, 2H, Arm. H, J = 8.0 Hz), 7.60 (t, 1H, Arm. H, J = 8.0 Hz), 7.71 (d, 1H, Arm. H, J = 8.0 Hz), 7.90 (d, 1H, Arm. H, J = 8.0 Hz), 8.40 (d, 1H, Arm. H, J = 8.0 Hz), 8.92 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.82, 116.64, 119.42, 120.68, 124.54, 124.72, 126.29, 129.11, 129.39 (2 C), 129.69 (2 C), 132.53, 132.71, 133.33, 136.59, 137.03, 143.42, 145.16, 151.89, 154.02, 156.88, 160.27, 194.30. Anal. calcd for C24H16ClNO3: C, 71.73; H, 4.01; N, 3.49. Found: C, 71.95; H, 3.97; N, 3.52.
4.1.3.4. 3-(6-Methyl-5-[(2E)-3-(4-methylphenyl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5d)
A yellow powder, yield: 71%. Mp: 206–208 °C. IR (νmax/cm−1): 3054 (CH-arom.), 2967, 2922 (CH-aliph.), 1727, 1661 (2 C = O). 1H NMR (400 MHz, CDCl3-d) δ: 2.42 (s, 3H, CH3), 2.78 (s, 3H, CH3), 7.13 (d, 1H, COCH=CH, J = 16.0 Hz), 7.25 (d, 1H, Arm. H, J = 8.0 Hz), 7.27 (d, 1H, Arm. H, J = 8.0 Hz), 7.37 (t, 1H, Arm. H, J = 8.0 Hz), 7.42 (d, 1H, Arm. H, J = 8.0 Hz), 7.51 (d, 1H, COCH = CH, J = 16.0 Hz), 7.52 (d, 2H, Arm. H, J = 8.0 Hz), 7.62 (t, 1H, Arm. H, J = 8.0 Hz), 7.73 (d, 1H, Arm. H, J = 8.0 Hz), 7.92 (d, 1H, Arm. H, J = 8.0 Hz), 8.42 (d, 1H, Arm. H, J = 8.0 Hz), 8.95 (s, 1H, 4-H of coumarin ring). 13C NMR (100 MHz, CDCl3-d) δ: 21.63, 116.50 (2 C), 119.15, 121.89, 124.72, 124.98 (2 C), 128.79 (2 C), 129.62, 129.93 (2 C), 131.28, 133.12, 134.59, 138.04, 142.17, 145.08, 147.84, 150.97, 154.22, 156.42, 159.94, 193.69. Anal. calcd for C25H19NO3: C, 78.72; H, 5.02; N, 3.67. Found: C, 79.02; H, 4.97; N, 3.65.
4.1.3.5. 3-(6-Methyl-5-[(2E)-3-(4-nitrophenyl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5e)
A red powder, yield: 57%. Mp: 202–204 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.74 (s, 3H, CH3), 7.14 (d, 1H, COCH=CH, J = 16.0 Hz), 7.36 (t, 1H, Arm. H, J = 8.0 Hz), 7.41 (d, 3H, Arm. H, J = 8.0 Hz), 7.48 (d, 1H, COCH = CH, J = 16.0 Hz), 7.53 (d, 2H, Arm. H, J = 8.0 Hz), 7.61 (t, 1H, Arm. H, J = 8.0 Hz), 7.71 (d, 1H, Arm. H, J = 8.0 Hz), 7.90 (d, 1H, Arm. H, J = 8.0 Hz), 8.41 (d, 1H, Arm. H, J = 8.0 Hz), 8.91 (s, 1H, 4-H coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.81, 116.43, 119.41, 120.67, 124.53, 124.72, 126.27, 129.10, 129.38 (2 C), 129.69 (2 C), 132.53, 132.69, 133.31, 136.59, 137.02, 143.43, 145.18, 151.88, 154.00, 156.87, 160.27, 194.31. Anal. calcd for C24H16N2O5: C, 69.90; H, 3.91; N, 6.79. Found: C, 70.11; H, 3.90; N, 6.83.
4.1.3.6. 3-(5-[(2E)-3-[4-(Dimethylamino)phenyl]prop-2-enoyl]-6-methylpyridin-2-yl)-2H-chromen-2-one (5f)
An orange powder, yield: 73%. Mp: 196–198 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.71 (s, 3H, CH3), 3.05 (s, 6H, N(CH3)2), 6.68 (d, 2H, Arm. H, J = 8.0 Hz), 6.92 (d, 1H, COCH=CH, J = 16.0 Hz), 7.34 (t, 1H, Arm. H, J = 8.0 Hz), 7.40 (d, 1H, Arm. H, J = 8.0 Hz), 7.41 (d, 1H, COCH = CH, J = 16.0 Hz), 7.27 (d, 2H, Arm. H, J = 8.0 Hz), 7.58 (t, 1H, Arm. H, J = 8.0 Hz), 7.70 (d, 1H, Arm. H, J = 8.0 Hz), 7.83 (d, 1H, Arm. H, J = 8.0 Hz), 8.35 (d, 1H, Arm. H, J = 8.0 Hz), 8.87 (s, 1H, 4-H coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.51, 40.07 (2 C), 111.79 (2 C), 116.37, 119.50, 120.62, 121.15, 121.75, 124.64, 124.87, 129.03, 130.65 (2 C), 132.29, 134.67, 136.22, 143.03, 148.28, 151.16, 152.34, 153.94, 156.30, 160.34, 195.20. Anal. calcd for C26H22N2O3: C, 76.08; H, 5.40; N, 6.82. Found: C, 75.83; H, 5.46; N, 6.84.
4.1.3.7. 3-(6-Methyl-5-[(2E)-3-[4-(morpholin-4-yl)phenyl]prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5g)
A yellow powder, yield: 60%. Mp: 208–210 °C. IR (νmax/cm−1): 3065 (CH-arom.), 2958, 2918 (CH-aliph.), 1727, 1656 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.72 (s, 3H, CH3), 3.28 (t, 4H, morpholinyl ring, J = 5.0 Hz), 3.86 (t, 4H, morpholinyl ring, J = 5.0 Hz), 6.88 (d, 2H, Arom. H, J = 8.0 Hz), 6.99 (d, 1H, COCH=CH, J = 16.0 Hz), 7.34 (t, 1H, Arom. H, J = 8.0 Hz), 7.40 (d, 1H, Arom. H, J = 8.0 Hz), 7.42 (d, 1H, COCH = CH, J = 16.0 Hz), 7.50 (d, 2H, Arom. H, J = 8.0 Hz), 7.59 (t, 1H, Arom. H, J = 8.0 Hz), 7.70 (d, 1H, Arom. H, J = 8.0 Hz), 7.85 (d, 1H, Arom. H, J = 8.0 Hz), 8.37 (d, 1H, Arom. H, J = 4.0 Hz), 8.88 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.59, 47.74 (2 C), 66.56 (2 C), 114.48 (2 C), 116.39, 119.47, 120.63, 122.80, 124.67, 124.75, 124.87, 129.05, 130.35 (2 C), 132.38, 134.23, 136.32, 143.15, 147.29, 151.38, 153.04, 153.96, 156.45, 160.31, 195.05. Anal. calcd for C28H24N2O4: C, 74.32; H, 5.35; N, 6.19. Found: C, 74.20; H, 5.37; N, 6.24.
4.1.3.8. 3-(5-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]-6-methylpyridin-2-yl)-2H-chromen-2-one (5h)
A yellow powder, yield: 66%. Mp: 174–175 °C. IR (νmax/cm−1): 3058 (CH-arom.), 2965, 2931 (CH-aliph.), 1725, 1660 (2 C = O). 1H NMR (400 MHz, CDCl3-d) δ: 2.77 (s, 3H, CH3), 3.88 (s, 3H, O CH3), 6.96 (d, 2H, Arom. H, J = 8.0 Hz), 7.05 (d, 1H, COCH=CH, J = 16.0 Hz), 7.37 (t, 1H, Arom. H, J = 8.0 Hz), 7.41 (d, 1H, Arom. H, J = 8.0 Hz), 7.48 (d, 1H, COCH = CH, J = 16.0 Hz), 7.57 (d, 1H, Arom. H, J = 8.0 Hz), 7.62 (t, 1H, Arom. H, J = 8.0 Hz), 7.74 (d, 1H, Arom. H, J = 8.0 Hz), 7.90 (d, 2H, Arom. H, J = 8.0 Hz), 8.42 (d, 1H, Arom. H, J = 8.0 Hz), 8.94 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.65, 55.43, 114.53 (2 C), 116.39, 119.44, 120.63, 123.87, 124.68 (2 C), 126.85, 129.07, 130.43 (2 C), 132.41, 133.94, 136.40, 143.24, 146.90, 151.53, 153.90, 156.55, 160.20, 162.08, 194.95. Anal. calcd for C25H19NO4: C, 75.55; H, 4.82; N, 3.52. Found: C, 75.38; H, 4.83; N, 3.54.
4.1.3.9. 3-(6-Methyl-5-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5i)
A yellow powder, yield: 86%. Mp: 186–188 °C. IR (νmax/cm−1): 3062 (CH-arom.), 2999, 2934, 2839 (CH-aliph.), 1727, 1666 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.72 (s, 3H, CH3), 3.90 (s, 9H, 3 of O CH3), 6.80 (s, 2H, Arom. H), 7.02 (d, 1H, COCH=CH, J = 16.0 Hz), 7.35 (t, 1H, Arom. H, J = 8.0 Hz), 7.40 (d, 1H, COCH = CH, J = 16.0 Hz), 7.41 (d, 1H, Arom. H, J = 8.0 Hz), 7.60 (t, 1H, Arom. H, J = 8.0 Hz), 7.71 (d, 1H, Arom. H, J = 8.0 Hz), 7.87 (d, 1H, Arom. H, J = 8.0 Hz), 8.38 (d, 1H, Arom. H, J = 8.0 Hz), 8.89 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.60, 56.17 (2 C), 61.01, 105.65 (2 C), 116.42, 119.41, 120.63, 124.65, 124.73, 125.63, 129.07, 129.55, 132.50, 133.58, 136.43, 140.75, 143.34, 147.27, 151.69, 153.49 (2 C), 153.96, 156.59, 160.36, 195.03. Anal. calcd for C27H23NO6: C, 70.89; H, 5.07; N, 3.06. Found: C, 71.07; H, 5.02; N, 3.08.
4.1.3.10. 3-(5-[(2E)-3-(2H-1,3-Benzodioxol-5-yl)prop-2-enoyl]-6-methylpyridin-2-yl)-2H-chromen-2-one (5j)
A green powder, yield: 84%. Mp: 178–180 °C. IR (νmax/cm−1): 3064 (CH-arm.), 2970, 2903 (CH-aliph.), 1723, 1657 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.72 (s, 3H, CH3), 6.03 (s, 2H, OCH2O), 6.84 (d, 1H, Arm. H, J = 8.0 Hz), 6.98 (d, 1H, COCH=CH, J = 16.0 Hz), 7.05 (d, 1H, Arm. H, J = 8.0 Hz), 7.10 (s, 1H, Arm. H), 7.33 (t, 1H, Arm. H, J = 8.0 Hz), 7.40 (d, 1H, Arm. H, J = 8.0 Hz), 7.42 (d, 1H, COCH = CH, J = 16.0 Hz), 7.60 (t, 1H, Arm. H, J = 8.0 Hz), 7.70 (d, 1H, Arm. H, J = 8.0 Hz), 7.86 (d, 1H, Arm. H, J = 8.0 Hz), 8.37 (d, 1H, Arm. H, J = 4.0 Hz), 8.89 (s, 1H, 4-H coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.70, 101.74, 106.61, 108.73, 116.39, 119.42, 120.64, 124.10, 124.62, 124.68, 125.63, 128.62, 129.07, 132.43, 133.81, 136.43, 143.27, 146.73, 148.51, 150.36, 151.60, 153.96, 156.62, 160.27, 194.66. Anal. calcd for C25H17NO5: C, 72.99; H, 4.16; N, 3.40. Found: C, 73.23; H, 4.14; N, 3.42.
4.1.3.11. 3-(6-Methyl-5-[(2E)-3-(naphthalen-1-yl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5k)
A yellow powder, yield: 72%. Mp: 204–206 °C. IR (νmax/cm−1): 3040 (CH-arom.), 2962, 2923 (CH-aliph.), 1721, 1660 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.82 (s, 3H, CH3), 7.29 (d, 1H, COCH=CH, J = 16.0 Hz), 7.35 (t, 1H, Arm. H, J = 8.0 Hz), 7.41 (d, 1H, Arm. H, J = 8.0 Hz), 7.53–7.60 (m, 4H, Arm. H), 7.72 (d, 1H, Arm. H, J = 8.0 Hz), 7.90 (d, 1H, Arm. H, J = 8.0 Hz), 7.95 (d, 2H, Arm. H, J = 8.0 Hz), 8.01 (d, 1H, Arm. H, J = 8.0 Hz), 8.12 (d, 1H, Arm. H, J = 8.0 Hz), 8.44 (d, 1H, COCH = CH, J = 16.0 Hz), 8.46 (d, 1H, Arm. H, J = 8.0 Hz), 8.94 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 24.00, 116.41, 119.42, 120.74, 123.08, 124.52, 124.70, 125.38, 125.46, 126.39, 127.20, 128.10, 128.86, 129.11, 131.35, 131.50, 131.55, 132.50, 133.58, 133.70, 136.74, 143.32, 143.42, 151.86, 154.00, 157.01, 160.26, 194.18. Anal. calcd for C28H19NO3: C, 80.56; H, 4.59; N, 3.36. Found: C, 80.70; H, 4.63; N, 3.33.
4.1.3.12. 3-(6-Methyl-5-[(2E)-3-(pyridin-2-yl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5l)
A yellow powder, yield: 80%. Mp: 188–190 °C. IR (νmax/cm−1): 3067 (CH-arom.), 2978, 2921 (CH-aliph.), 1725, 1663 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.77 (s, 3H, CH3), 7.29 (t, 1H, Arom. H, J = 8.0 Hz), 7.32 (q, 1H, Arom. H, J = 8.0 Hz), 7.40 (d, 1H, Arom. H, J = 8.0 Hz), 7.49 (d, 1H, Arom. H, J = 8.0 Hz), 7.54 (d, 1H, COCH=CH, J = 16.0 Hz), 7.58 (d, 1H, Arom. H, J = 8.0 Hz), 7.69 (d, 1H, Arom. H, J = 8.0 Hz), 7.70 (d, 1H, COCH = CH, J = 16.0 Hz), 7.75 (t, 1H, Arom. H, J = 8.0 Hz), 8.01 (d, 1H, Arom. H, J = 8.0 Hz), 8.41 (d, 1H, Arom. H, J = 8.0 Hz), 8.68 (d, 1H, Arom. H, J = 4.0 Hz), 8.91 (s, 1H, 4-H coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 24.02, 116.40, 119.42, 120.69, 124.69, 124.52, 124.68, 124.71, 125.15, 129.00, 129.11, 132.48, 133.14, 136.92, 136.96, 143.43, 144.54, 150.31, 151.94, 152.73, 154.01, 157.18, 194.19. Anal. calcd for C23H16N2O3: C, 74.99; H, 4.38; N, 7.60. Found: C, 75.21; H, 4.40; N, 7.54.
4.1.3.13. 3-(6-Methyl-5-[(2E)-3-(thiophen-2-yl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5m)
A yellow powder, yield: 71%. Mp: 197–199 °C. IR (νmax/cm−1): 3055 (CH-arom.), 3001, 2930 (CH-aliph.), 1722, 1659 (2 C = O). 1H NMR (500 MHz, CDCl3-d) δ: 2.73 (s, 3H, CH3), 6.95 (d, 1H, COCH=CH, J = 16.0 Hz), 7.09 (t, 1H, Arm. H, J = 4.0 Hz), 7.33–7.40 (m, 3H, Arm. H), 7.47 (d, 1H, Arm. H, J = 4.0 Hz), 7.59 (t, 1H, Arm. H, J = 8.0 Hz), 7.65 (d, 1H, COCH = CH, J = 16.0 Hz), 7.69 (d, 1H, Arm. H, J = 8.0 Hz), 7.88 (d, 1H, Arm. H, J = 8.0 Hz), 8.39 (d, 1H, Arm. H, J = 8.0 Hz), 8.89 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 23.72, 116.37, 119.39, 120.64, 124.56, 124.66, 128.50, 129.06, 129.83, 132.43, 132.55, 133.48, 136.45, 137.85, 138.99, 139.62, 143.29, 151.70, 153.95, 156.75, 160.23, 193.96. Anal. calcd for C22H15NO3S: C, 70.76; H, 4.05; N, 3.75. Found: C, 70.89; H, 4.02; N, 3.76.
4.1.3.14. 3-(6-Methyl-5-[(2E)-3–(5-methylfuran-2-yl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5n)
A yellow powder, yield: 58%. Mp: 188–190 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.38 (s, 3H, CH3), 2.74 (s, 3H, CH3), 6.14 (s, 1H, Arm. H), 6.64 (s, 1H, Arm. H), 6.96 (d, 1H, COCH=CH, J = 16.0 Hz), 7.26 (d, 1H, COCH = CH, J = 16.0 Hz), 7.34 (t, 1H, Arom. H, J = 8.0 Hz), 7.40 (d, 1H, Arom. H, J = 8.0 Hz), 7.59 (t, 1H, Arom. H, J = 8.0 Hz), 7.70 (d, 1H, Arom. H, J = 8.0 Hz), 7.90 (d, 1H, Arom. H, J = 8.0 Hz), 8.37 (d, 1H, Arom. H, J = 8.0 Hz), 8.89 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 14.04, 23.63, 109.67, 116.40, 119.28, 119.41, 120.80, 121.32, 124.70, 129.11, 129.24, 132.49, 133.97, 136.58, 137.74, 143.39, 149.55, 151.47, 153.97, 156.71, 156.83, 160.27, 193.92. Anal. calcd for C23H17NO4: C, 74.38; H, 4.61; N, 3.77. Found: C, 74.51; H, 4.61; N, 3.80.
4.1.3.15. 3-(6-Methyl-5-[(2E)-3–(3-methyl-1-phenyl-1H-pyrazol-4-yl)prop-2-enoyl]pyridin-2-yl)-2H-chromen-2-one (5o)
A yellow powder, yield: 61%. Mp: 204–206 °C. 1H NMR (500 MHz, CDCl3-d) δ: 2.48 (s, 3H, CH3), 2.75 (s, 3H, CH3), 6.94 (d, 1H, COCH=CH, J = 16.0 Hz), 7.30–7.35 (m, 2H, Arm. H), 7.40 (d, 1H, Arm. H, J = 8.0 Hz), 7.46 (t, 2H, Arm. H, J = 8.0 Hz), 7.52 (d, 1H, COCH = CH, J = 16.0 Hz), 7.59 (t, 1H, Arm. H, J = 8.0 Hz), 7.67 (d, 2H, Arm. H, J = 8.0 Hz), 7.70 (d, 1H, Arm. H, J = 8.0 Hz), 7.88 (d, 1H, Arm. H, J = 8.0 Hz), 8.14 (s, 1H, Arm. H), 8.39 (d, 1H, Arm. H, J = 8.0 Hz), 8.90 (s, 1H, 4-H of coumarin ring). 13C NMR (125 MHz, CDCl3-d) δ: 13.31, 23.76, 116.40, 118.12, 119.11 (2 C), 119.43, 120.67, 124.15, 124.62, 124.69, 127.08, 127.79, 129.07, 129.56 (2 C), 132.44, 133.80, 136.40, 137.20, 139.20, 143.27, 150.91, 151.61, 153.98, 156.69, 160.28, 194.39. Anal. calcd for C28H21N3O3: C, 75.15; H, 4.73; N, 9.39. Found: C, 75.32; H, 4.75; N, 9.44.
4.2. Biological evaluation
4.2.1. Carbonic anhydrase isoforms inhibition assay
The CA inhibition activity for the herein reported MPC derivatives was evaluated against the hCA isoforms I, II, IX, and XII using stopped-flow CO2 hydrase testCitation7,Citation33–36 (see the Supplementary Material).
4.2.2. In vitro antitumor screening against 59 cancer cell lines
The anticancer test was conducted using the methods of the Drug Evaluation Branch, National Cancer Institute, Bethesda, MD, using 59 human tumour cell lines derived from nine human tissues. The GI50, TGI, and LC50 dose-response parameters were calculated for each medicationCitation37–39.
Supplemental Material
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CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
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References
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