Novel coumarins and benzocoumarins acting as isoform-selective inhibitors against the tumor-associated carbonic anhydrase IX

Abstract

A series of coumarins and benzocoumarins incorporating methyl and hydroxyl moieties in the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and to a slightly less extent, CA XII. The nature and position of the groups substituting the coumarin ring influenced CA inhibitory properties. 4-Methyl-5,7-dihydroydroxycoumarin showed K_Is >200 µM against CA I and II, of 0.19 µM against CA IX and of 6.4 µM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads for designing isoform-selective enzyme inhibitors.

• Benzocoumarin
• carbonic anhydrase
• coumarin
• hydroxycoumarin
• selective enzyme inhibitor
• tumor-associated isoforms IX and XII

Introduction

Coumarins are a class of widely spread natural compounds which was only recently revealed to inhibit the zinc enzyme carbonic anhydrase CA (EC 4.2.1.11)1–3. CAs are ubiquitous enzymes in organisms throughout the tree of life, with several genetically distinct families encoding them in prokaryotes and eukaryotes4–11. CAs are inhibited by metal complexing anions, sulfonamides and their isosteres (sulfamates, sulfamides, etc.), phenols and polyamines, which bind either to the metal ion within the enzyme active site or are anchored to the water molecule coordinated to it11. However, the coumarin inhibition mechanism of the α-CAs is totally different and was only recently deciphered1,2. Coumarin derivatives were recently shown to constitute a totally new class and type of CA inhibitors (CAIs), being “prodrugs” which are hydrolyzed within the CA active site with formation of 2-hydroxycinnamic acids.

The coumarins represent an interesting class of CAIs due to several reasons:

1. The inhibition mechanism is different from those of other CAIs, and due to their binding at the entrance of the enzyme active site, where there is a rather large variability in the 3D structure and amino acid sequence between the various isoforms, may lead to compounds with isoform-selective behavior1–3.

2. Coumarins are widespread natural products and are also easily prepared synthetically with a large range of substitution patterns, incorporating moieties of diverse chemical nature, in different numbers of such moieties and in diverse positions of the coumarin ring.

A natural product coumarin, 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one, isolated from the Australian plant Leionema ellipticum, was the first compound shown to possess significant CA inhibitory activity1. By means of X-ray crystallography of it’s adduct with the human (h) isoform hCA II, it was evidenced the presence of a substituted 2-hydroxy-cinnamic acid in the enzyme active site, which is the hydrolysis product (mediated by the CA) of the original coumarin1. The same situation has been thereafter observed for the simple, unsubstituted coumarin2. The 2-hydroxy-cinnamic acids formed from the original coumarins occluded the entrance to the enzyme active site, a mechanism never evidenced before for CA inhibition2,11. This region of the CA active site is on the other hand the most variable one among the 16 CA isoforms present in mammals, and this explains the observations that many coumarin derivatives show a high selectivity ratio for inhibiting various CA isoforms, many of which have pharmacologic applications1–3.

In fact recently, some glycosyl-substituted coumarins with potent inhibitory activity and selectivity for the tumor-associated isozyme CA IX and XII, were shown to strongly inhibit the growth of primary tumors and metastases overexpressing these enzymes, in an animal model of breast cancer3. For this reason, exploring novel coumarin derivatives as CAIs, or compounds known in the literature but not yet investigated for this activity, is of interest in the design of isoform-selective pharmacological agents of this type. Indeed, CAIs have a range of applications as antiglaucoma, antiobesity, antiepileptic or diuretic agents12–26. Furthermore, inhibition of CAs from various bacteria or fungi may also lead to the development of antiinfectives based on CAIs27,28. Here, we report the synthesis of several coumarin/benzocoumarin derivatives and their inhibition against medicinally important CA isoforms, such as CA I, II, IX and XII.

Materials and methods

Chemistry

The Pechmann condensation is the most widely used method for coumarin synthesis, as it uses simple starting materials and gives good yields for variously substituted coumarins29,30. The Pechmann condensation allows the synthesis of coumarins by reaction of phenols with β-keto esters. The reaction was carried out by addition of TiCl₄ to the mixture of the reagents, with stirring as exemplified below for the preparation of coumarins/benzocoumarins 1–5.

Synthesis of 7-hydroxy-4-methyl-2H-2-one 1

Resorcinol (10 mmol, 1.10 g) and ethyl acetoacetate (20 mmol, 2.5 ml) were mixed thoroughly in round bottom flask and titanium (IV) chloride (10 mmol) was added to the mixture with constant stirring at room temperature. Small amount of ethanol was added, to facilitate the stirring of mixture. The reaction was completed in 5–6 h. The thin layer chromatography was performed to check the completion of reactions. On the completion of reactions as indicated by TLC, solvent was evaporated and reaction mixture was poured onto crushed ice and the resulting crude product was filtered off and recrystallized from ethanol–water mixture (1:1) and then, purified by column chromatography. Yield 78%; Rf: 0.428 (ethyl acetate/hexane 3:7); m.p.: 188–190 °C; ¹HNMR (400 MHz, DMSO): δ ppm 2.257 (s, 3H, Me), 6.001(s, 1H, C=CH), 6.626–7.439 (m, 3H, ArH), 10.463 (s, 1H, ArOH); IR (KBr) 3491, 3123, 2949, 1672, 1672, 1601, 1272 cm⁻¹. EIMS: m/z: 176 (M+).

Synthesis of 5,7-dihydroxy-4-methyl-2H-chromen-2-one 2

Phloroglucinol (10 mmol, 1.26 g) and ethyl acetoacetate (20 mmol, 2.5 ml) were mixed thoroughly in round bottom flask and titanium (IV) chloride (10 mmol) was added to the mixture with constant stirring at room temperature. Small amount of ethanol was added, to facilitate the stirring of mixture. The reaction was completed in 18–20 h. The thin layer chromatography was performed to check the completion of reactions. On the completion of reactions as indicated by TLC, solvent was evaporated and reaction mixture was poured onto crushed ice and the resulting crude product was filtered off and recystallized from ethanol–water mixture (1:1) and purified by column chromatography. Yield 70%; Rf: 0.269 (ethyl acetate/hexane 3:7); m.p.: 260–262 °C; ¹HNMR (400 MHz, DMSO): δ ppm 2.503 (s, 3H, Me), 5.848(s, 1H, C=CH), 6.162–6.255 (d, 2H ArH), 10.297 (s, 1H, ArOH), 10.523 (S, 1H, ArOH); IR (KBr) 3421, 3124, 2922, 1663, 1618, 1296 cm⁻¹; EIMS: m/z: 192 (M+).

Synthesis of 7,8-dihydroxy-4-methyl-2H-chromen-2-one 3

Pyrogallol (benzene-1,2,3-triol, 10 mmol) and ethyl acetoacetate (20 mmol) were mixed thoroughly in round bottom flask and titanium (IV) chloride (10 mmol) was added to the mixture with constant stirring at room temperature. Small amount of ethanol was added, to facilitate the stirring of mixture. The reaction was completed in 8–9 h. The thin layer chromatography was performed to check the completion of reactions. On the completion of reactions as indicated by TLC, solvent was evaporated and reaction mixture was poured onto crushed ice and the resulting crude product was filtered off and recystallized from ethanol–water mixture (1:1) and purified by column chromatography. Yield 72%; Rf: 0.283 (ethyl acetate/hexane 3:7); m.p.: 260–262 °C; ¹HNMR (400 MHz, DMSO): δ ppm 2.339 (s, 3H, Me), 6.108 (s, C=CH), 6.783–7.090 (m, 2H, ArH), 9.644 (s, 1H, ArOH); IR (KBr) 3412, 3233, 2865, 1663, 1609, 1597, 1236 cm⁻¹; EIMS: m/z: 192 (M+).

Synthesis of 4-methyl-2H-benzo[h]chromen-2-one 4

α-Naphthol (10 mmol) and ethyl acetoacetate (20 mmol) were mixed thoroughly in round bottom flask and titanium (IV) chloride (10 mmol) was added to the mixture with constant stirring at room temperature. Small amount of ethanol was added, to facilitate the stirring of mixture. The reaction was completed in 3–4 h. The thin layer chromatography was performed to check the completion of reactions. On the completion of reactions as indicated by TLC, solvent was evaporated and reaction mixture was poured onto crushed ice and the resulting crude product was filtered off and recystallized from ethanol–water mixture (1:1) and purified by column chromatography. Yield 60%; Rf: 0.352 (ethyl acetate/hexane 3:7); m.p.: 154–156 °C; ¹HNMR (400 MHz, DMSO): δ ppm 1.827 (s, 3H, Me), 6.464 (s, 1H, C=CH), 7.694–8.326 (m, 6H, ArH)); IR (KBr) 3059, 2920, 1707, 1609, 1273 cm⁻¹.; EIMS: m/z: 210 (M+).

Synthesis of 4-methyl-2H-benzo[g]chromen-2-one 5

β-Naphthol (10 mmol) and ethyl acetoacetate (20 mmol) were mixed thoroughly in round bottom flask and titanium (IV) chloride (10 mmol) was added to the mixture with constant stirring at room temperature. Small amount of ethanol was added, to facilitate the stirring of mixture. The reaction was completed in 3–4 h. The thin layer chromatography was performed to check the completion of reactions. On the completion of reactions as indicated by TLC, solvent was evaporated and reaction mixture was poured onto crushed ice and the resulting crude product was filtered off and recystallized from ethanol–water mixture (1:1) and purified by column chromatography. Yield 81%; Rf: 0.403 (ethyl acetate/hexane 3:7); m.p.: 150–150 °C; ¹HNMR (400 MHz, DMSO): δ ppm 1.689 (s, 3H, Me), 7.052–7.749 (m, 6H, ArH); IR (KBr) 3273, 2866, 1674, 1630, 1277 cm⁻¹.; EIMS: m/z: 210 (M+).

CA inhibition

An applied photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO₂ hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants31. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min to 72 h at room temperature (15 min) or 4 °C (all other incubation times) prior to assay, in order to allow for the formation of the E-I complex or for the eventual active site mediated hydrolysis of the inhibitor. Data reported in Table 1 show the inhibition after 6 h incubation, which led to the completion of the in situ hydrolysis of the coumarin and formation of the 2-hydroxy-cinnamic acids. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier1–3, and represent the mean from at least three different determinations. CA isoforms were recombinant ones obtained in house as reported earlier1–3.

Table 1. hCA I, II, IX and XII inhibition data with coumarins 1–5 by a stopped flow, CO₂ hydrase assay31.

		Ki (µM)*
Compounds	hCA I	hCA II	hCA IX	hCA XII
1	4.8	>200	0.45	4.3
2	8.4	>200	0.19	6.4
3	8.7	>200	0.27	6.6
4	9.0	>200	0.23	5.5
5	8.2	>200	0.20	3.3

*Mean from three different assays, errors in the range of ±10% of the reported values.

Discussion and conclusions

In previous contributions from our laboratory, we showed that even simple coumarins derivatives may lead to interesting inhibition profiles against various CA isoforms, with the discovery of a wide range of isoform-selective CAIs1,2,29,30. Thus, we investigated here the synthesis of new coumarins and inhibition of several pharmacologically important CA isoforms (e.g. CA I, II, IX and XII) with a small series of such compounds, i.e. coumarins (1–3) and benzocoumarins (4 and 5) (Scheme 1).

Scheme 1. Preparation of the coumarins/benzocoumarins 1–5 from phenols A, C and D and ethyl acetoacetate B.

Compounds 1–5 were prepared by literature procedures, by the Pechmann condensation of phenols A or naphthols C and D, with ethyl acetoacetate B, in the presence of Ti(IV) chloride as catalyst29,30. Compounds 1–5 have been characterized by standard physico–chemical procedures which confirmed their structures (“Materials and methods” section for details).

Coumarins 1–5 were investigated as inhibitors31 of four catalytically active hCA isoforms, hCA I, hCA II (cytosolic isoforms) as well as hCA IX and hCA XII (transmembrane, tumor-associated enzymes)32 (Table 1). The following structure–activity relationship (SAR) can be evidenced from the data of Table 1:

1. The slow cytosolic isoform hCA I was moderately inhibited by coumarins 1–5, with inhibition constants in the range of 4.8–9.0 µM. The bets inhibitor was 4-methyl-7-hydroxy-coumarin 1 (K_I of 4.8 µM), whereas the remaining 4 derivatives showed a flat, less potent inhibition (K_Is around 9 µM, Table 1).

2. As for other simple coumarins investigated earlier29,30, also coumarins 1–3 and benzocoumarins 4 and 5 were devoid of inhibitory activity against the dominant isoform hCA II, which is a very interesting feature of this class of CAIs. Indeed, the house-keeping, and quite abundant isoform hCA II showed K_Is > 200 µM with all these compounds, which constitutes a highly attractive feature for inhibitors that should target isoforms involved in tumorigenesis (CA IX and XII)32 or in other processes, such as for example obesity (CA VA and VB)33,34.

3. The tumor-associated hCA IX was well-inhibited by compounds 1–5, with K_Is in the submicromolar range, i.e. of 0.19–0.45 µM (Table 1). The best inhibitors were the linear benzocoumarin 5 (K_I of 0.20 µM) and the dihydroxy-4-methyl-coumarin 2 (K_I of 0.19 µM), but the remaining derivatives were only slightly less effective CAIs than 2 and 5.

4. The second tumor-associated isoform, hCA XII, was also inhibited by these compounds but less efficiently compared to hCA IX. Indeed, the coumarins/benzocoumarins 1–5 showed inhibition constants in the range of 3.3–6.6 µM. Again thus, a quite compact behavior, with little SAR, which may be explained by the rather similar structures of the investigated compounds.

In conclusion, a small series of coumarins/benzocoumarins incorporating methyl and hydroxyl moieties in the heterocyclic ring were investigated as CAIs. These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and XII. The nature and position of the groups substituting the coumarin ring influenced CA inhibitory properties. 5,7-Dihydroydroxycoumarin showed K_Is > 200 µM against CA II, of 0.19 µM against CA IX and of 6.4 µM against CA XII, being thus an isoform-selective, efficient inhibitor for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads for designing isoform-selective enzyme inhibitors.

Declaration of interest

The authors report no conflict of interest. This work was supported by an EU FP7 research grant (Metoxia project).

Acknowledgements

We are grateful to Dr Alfonso Maresca and Dr Daniela Vullo for technical support.