Novel 3-(6-methylpyridin-2-yl)coumarin-based chalcones as selective inhibitors of cancer-related carbonic anhydrases IX and XII endowed with anti-proliferative activity

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.


Introduction
Carbonic anhydrases (CAs) are vital for the processes of CO 2 hydration and HCO 3 dehydration 1-3 . The a-CAs are one of the seven known CAs families which are predominantly found in vertebrates, green plants cytoplasm, bacteria, and algae 4,5 . 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 approach 4,6 . 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 malignancies [7][8][9][10] . Furthermore, non-selective inhibition of hCAs leads to some side effects while treating cancer 11 . 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 ZBG 11,12 . 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 zinc 13,14 .
Coumarin ring, as a privileged scaffold, exerted exceptional anticancer profile acting through various mechanisms of action 15,16 . Coumarin (I, Figure 1) derivatives were introduced by Supuran' group as a non-classical type of CAIs 17 . Coumarin was shown to undergo hydrolysis to form cis-2-hydroxy-cinnamic acid (II, Figure 1), 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 isoforms 12,17,18 . These findings grasped the attention for developing a variety of coumarin-based CAIs, such as compounds III-V (Figure 1), which exerted efficient and selective inhibition activity towards the cancer-related isozymes IX and XII over the constitutional isozymes CA I and II [18][19][20] .
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 imatinib 21 , sorafenib 22 , and acalabrutinib 23 . The pyridine-based small molecules bearing chalcone functionality VI-VIII (Figure 1) have been described for their in vitro anticancer activity against different cancer cell lines [24][25][26][27] . In addition, the pyridine derivatives VIII and IX were able to inhibit the cancer-related CA IX isoform selectively 24-27 . 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 (Figure 1). 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, Figure 1). 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.

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 method 28 . 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.
Sixteen compounds were synthesised in this study, and their structures were confirmed by using IR, 1 H NMR, and 13 C 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 (-CH 3 ), methylene (-CH 2 -), and methoxy (-OCH 3 ) group signals appeared in the aliphatic region for both protons and carbons spectra of the corresponding targets.

Carbonic anhydrase isoforms inhibition assay
The newly synthesised MPCs (3 and 5a-o) were assessed for their CA inhibition activity employing the stopped-flow CO 2 hydrase assay 29 for constitutional hCA (I/II) isoforms and cancer-linked hCA (IX/XII) isoforms. Inhibition values given in Table 1 revealed that the herein-reported MPCs have varying degrees of inhibitory action against the examined CA isoforms.
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 > CH 3 > Cl > N(CH 3 ) 2 > OCH 3 > NO 2 , 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 ¼ 12.0 and 27.4 mM, respectively) in comparison to the unsubstituted phenyl-bearing analogue 5a (KIs ¼ 1.5 mM).
It is worth mentioning that incorporation of an unsubstituted phenyl moiety led to MPC 5a with moderate hCA XII inhibitory action (KI ¼ 5.1 mM), whereas grafting a halogen like para-fluoro (MPC 5b) and para-chloro (MPC 5c) improved the inhibitory activity (KIs ¼ 2.7 and 1.9 mM, 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 ¼ 1.8 and 2.8 mM, respectively) in comparison to the unsubstituted phenylbearing counterpart MPC 5a (KI ¼ 5.1 mM). 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 ¼ 0.92, 2.7, and 1.9 mM, respectively). On the other hand, replacement of the phenyl moiety with the fused naphthyl carbocycle (MPC 5k; KI ¼ 21.4 mM) or the bulky 3-methyl-1-phenyl-pyrazole heterocycle (MPC 5o; KI ¼ 17.8 mM) 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 ¼ 0.95 and 0.68 mM, 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 mM. Accordingly, all the designed MPCs showed excellent selectivity towards both cancer-related isoform IX and XII, compared with the cytosolic isoforms (Table 2). 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.

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) 30 .

Preliminary single (10 mM) dose screening
The antiproliferative activities of MPC 3 and MPCs 5a-o were first evaluated in a 10 mM 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 antiproliferative activities towards different cancer cell lines (mean% GI ¼ 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.
Assessing the obtained GI % values (Table 3) 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.
NCI screening results for MPC 5g showed anti-proliferative activity against 31 human cancer cell lines indicating a broadspectrum activity. Compound 5g exerted its lethal action towards  (Table 3).

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 (Table 3). MPC 5l was then chosen for additional biological evaluation in a five-dose (0.01-100 mM) experiment. MPC 5l's GI 50 , TGI, and LC 50 response parameters were obtained for each of the cancer cell lines studied. TGI represents cytostatic impact, whereas GI 50 values reflect the extent of growth inhibitory effect. Furthermore, the LC 50 parameter is regarded as the cytotoxicity parameter for the hybrid under investigation.

Conclusions
In brief, the present study demonstrates the design and synthesis of novel 6-(methylpyridin-2-yl)-coumarins MPC 3 and MPC (5a-o)  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 GI 50 values ranging from 3.20 to 8.49 mM.

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 F 254 ) 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). 1 H and 13 C NMR spectra were carried out utilising the Bruker instrument at 400-500 MHz for 1 H NMR and at 100-125 MHz for 13 C NMR spectrophotometer, TMS is being used as an internal standard and chemical shifts were recorded in ppm on the d scale using CDCl 3 -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.

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.