Benzenesulfonamide decorated dihydropyrimidin(thi)ones: carbonic anhydrase profiling and antiproliferative activity

Abstract

In the last decades, carbonic anhydrases (CAs) have become the top investigated innovative pharmacological targets and, in particular, isoforms IX and XII have been widely studied due to the evidence of their overexpression in hypoxic tumors. The frantic race to find new anticancer agents places the quick preparation of large libraries of putative bioactive compounds as the basis of a successful drug discovery and development programme. In this context, multi-component and, in general, one-step reactions are becoming very popular and, among them, Biginelli's reaction gave clean and easy-to-isolate products. Thus, we synthesized a series of Biginelli's products (10–17a–b) and similar derivatives (20–21) bearing the benzenesulfonamide moiety, which is known to inhibit CA enzymes. Through the stopped-flow technique, we were able to assess their ability to inhibit the targeted CAs IX and XII in the nanomolar range with promising selectivity over the physiologically relevant isoforms I and II. Crystallography studies and docking simulations helped us to gain insight into the interaction patterns established in the enzyme–inhibitor complex. From a chemical similarity-based screening of in-house libraries of compounds, a diphenylpyrimidine (23) emerged. The surprisingly potent inhibitory activity of 23 for CAs IX and XII along with its strong antiproliferative effect on two (triple-negative breast cancer MDA-MB-231 and glioblastoma U87MG) cell lines laid the foundation for further investigation, again confirming the key role of CAs in cancer.

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Introduction

Cancer is one of the leading causes of mortality worldwide^1,2 and a disease that mostly impacts negatively on the quality of life of the patients and their families along with the public costs.³ The entire scientific community, both the pharma industry and academia, is frantically searching for new therapeutical options through the application of recently developed technologies, e.g., CRISPR gene editing,^4,5 robotic surgery,⁶ personalized medicine,⁷ and the generation of new drugs from chemical, natural, and biotechnological sources.⁸ Undoubtedly, the most chosen approach is based on the discovery and validation of new putative pharmacological targets, e.g. enzymes, receptors, and microRNAs,⁹ through a complex multidisciplinary study, often including artificial intelligence,¹⁰ in order to escape (multi)drug-resistance phenomena and reduce the risk of collateral effects that lower significantly the patient compliance.

In the last decades, some of us shed light on the roles and functions of the superfamily of carbonic anhydrases (CAs, EC 4.2.1.1) in relevant physiological processes in prokaryotes and eukaryotes by means of the reversible hydration of carbon dioxide (CO₂).¹¹ Eight genetic families (α, β, δ, ε, ζ, η, θ, and ι) have been reported to date, with human (h) genome encoding for 15 isoforms belonging to the α-class.¹² Their druggability has been already proved and several modulators, both inhibitors and activators,¹¹ have been developed, with some compounds reaching (pre)clinical use, such as diuretics, anti-glaucoma, anti-obesity, and anti-epileptic agents,¹³ owing to the smart tail approach.^14,15 In this context, the transmembrane hCAs IX and XII could be listed as the most promising anticancer targets.^16–21 These isoforms are overexpressed in several solid tumors, e.g., breast, lung, and colorectal, by the hypoxia-inducible factors (HIF-1α and -2α), allowing the maintenance of the optimal pH for cell survival in hypoxic conditions and also contributing to cancer cell migration, invasion, and stemness.¹⁶ Selective inhibition of the two isoenzymes was reported to impair their functionality, causing the cancer cell to undergo apoptosis in vitro on a large panel of cancer cell lines^22–24 and in vivo in xenograft tumor models.^25,26 For instance, a small molecule containing the prototypic benzenesulfonamide CA inhibitor SLC-0111 (Fig. 1) was developed and is currently under clinical trial in combination with gemcitabine for metastatic pancreatic ductal adenocarcinoma.^27,28

Fig. 1 Structure of licensed SLC-0111.

Moreover, the continuous race to quickly find new active molecules with chemical diversity leads researchers to generate large libraries through easy synthetic schemes, often by just one-step procedures. In particular, multi-component reactions, such as Biginelli,²⁹ could represent a good strategy that allows practically performing small chemical changes, employing just one different chemical per time, and, thereby, conducting easily but robust structure–activity relationships (SARs).^30–34 Indeed, Biginelli's reaction permits the high-yielding obtainment of 3,4-dihydropyrimidin-2-(1H)-ones and -thiones (DHPMs)³⁵ by reacting the suitable urea or thiourea with an aromatic aldehyde and a β-ketocarboxyl derivative.

Results

Chemistry

DHPMs 10–17a–b were synthesized through Biginelli's three-component reaction, as depicted in Scheme 1, by using 4-formyl benzenesulfonamide 1 (previously prepared as reported in Scheme S1 in ESI†),^36,37 the suitable active methylene-containing derivatives 2–9, and urea or thiourea in the presence of sodium hydrogen sulfate (NaHSO₄).³⁸

Scheme 1 Synthetic pathway for the target compounds 10–17a–b, 20 and 21. Reagents and conditions: i) urea (for compounds 10–17a) or thiourea (for compounds 10–12b and 14–17b), NaHSO₄, hexane/ACN, reflux, 6–24 h; ii) NaOH aq., EtOH, 0 °C–r.t., 16 h.

Compounds 2–6 were commercially available, while 7–9 were prepared by us, as reported in ESI† (Schemes S2 and S3).^39,40 Instead, pyrimidintriones 20–21 were obtained via condensation (Scheme 1), by reacting aldehyde derivative 1 with compounds 18 and 19.

Compound 23 was obtained in a straightforward synthetic pathway by first reacting intermediate 1 with acetophenone to afford chalcone 22, which in turn was cyclized with guanidine and oxidated by 30% H₂O₂ aq. through a two-step-one-pot reaction (5) (Scheme 2).

Scheme 2 Synthesis of 2-aminopyrimidine 23. Reagents and conditions: i) acetophenone, NaOH aq., EtOH, 0 °C–r.t., 16 h; ii) guanidine hydrochloride, EtOH, KOH aq.; then, 30% H₂O₂ aq., refl., 16 h.

Enzymatic evaluation on human carbonic anhydrases

The inhibition profiles of 10–12a–b, 13a, 14–17a–b, 20, 21, and 23 on the abundantly expressed hCAs I and II, the central nervous system (CNS)-associated hCA VII, and the hypoxic-tumor-associated hCAs IX and XII were determined through the stopped-flow CO₂ hydrase assay.⁴¹ Inhibition constants (K_I) are reported in Table 1 with the reference compound acetazolamide (AAZ).

Table 1 Inhibition data of sulfonamides 10–12a, 12b, 13a, 14–17a–b, 20, 21, 23 and reference compound AAZ on hCAs I, II, VII, IX, and XII through the stopped-flow CO₂ hydrase assay⁴¹

Compounds	K I (nM)^a
	hCA I	hCA II	hCA VII	hCA IX	hCA XII
a Mean from 3 different assays by a stopped-flow technique (errors were in the range of ±5–10% of the reported values).
10a	362	45.9	19.7	39.0	7.7
10b	91.4	31.4	63.8	8.1	9.8
11a	265	5.6	12.3	7.9	10.6
11b	116	0.29	14.5	9.9	9.2
12a	68.0	65.3	25.7	8.9	61.7
12b	286	0.86	15.5	8.1	33.6
13a	630	43.8	18.5	3.5	8.5
14a	16.1	2.1	10.1	36.0	594
14b	643	91.0	191	4.8	89.7
15a	272	16.6	102	30.5	289
15b	95.0	58.2	168	52.2	72.0
16a	22.8	8.4	25.0	7.3	9.7
16b	760	25.2	17.8	56.5	34.1
17a	217	70.5	88.0	6.6	8.9
17b	491	200	18.1	41.3	8.7
20	957	29.1	202	7.1	57.8
21	97.0	17.3	9.1	8.2	100
23	621	377	433	8.9	5.3
AAZ	250	12.1	2.5	25.8	5.7

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Overall, inhibition data in Table 1 showed that most of the obtained derivatives were remarkably selective for hCAs IX and XII, resulting in nanomolar inhibitors. In some cases, subnanomolar K_I values were found for hCA II (i.e., 11b and 12b). The following SARs are reported for hCAs I and II and tumoral isoforms IX and XII, while a separate section is dedicated to data on the VII isoenzymes.

The introduction of the isosteric sulfur atom within the 5-acetyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-4-yl moiety in 10a to afford 10b improved the inhibitory activity towards the isoforms I, II and IX by 4.0-, 1.5- and 4.8-fold, respectively, while a slight (1.3-fold) K_I value increase was noticed for the other tumor-associated isoform.

A different kinetic pattern was observed when the same substitution was performed on the 5-acetyl-6-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidin-4-yl moiety in 11a. In this case, the thiooxo derivative 11b was a more potent inhibitor than its parent compound only against hCAs I and II (by 2.3- and 19.3-fold, respectively), whereas very slight K_I variations were registered against the other isoforms.

As for the ethyl ester 12a, a consistent increase of the inhibitory potency was observed only for hCAs II and XII by 76- and 1.8-fold, respectively, while the K_I value for hCA I was enhanced by 4.2-fold. Quite interestingly, hCA IX was not affected by the O-S substitution (K_Is of 8.9 and 8.1 for 12a and 12b, respectively). The same isosteric substitution was explored for 14–17a–b and different SAR considerations could be generated.

For instance, the replacement of the methyl group at position 6 in the tetrahydropyrimidine ring in 12a with a phenyl group (14a) induced a relevant increase in the inhibitory potency for hCAs I and II by 4.2- and 31.1-fold, respectively (K_Is = 16.1 and 2.1 nM). Conversely, the tumor-associated IX and XII isoforms resulted in a far weak inhibition by 4.0- and 9.6-fold, respectively (K_Is of 36.0 and 594 nM). Interestingly, the thiooxo derivative of 14a, namely 14b, induced an opposite kinetic pattern with the K_I values for hCAs I and II of 643 and 91.0 nM, respectively, thus with a 39.9- and 43.3-fold increase. On the other hand, hCAs IX and XII resulted to be 7.5- and 6.6-fold more potently inhibited (K_Is of 4.8 and 89.7 nM), respectively.

The introduction of the fluorine and chlorine atoms in 14a to afford 15a and 16a, respectively, clearly induced substantial changes in the in vitro kinetics. Data in Table 1 showed that the fluoro raised the K_I values for hCAs I and II by 16.9- and 7.9-fold, respectively, and, conversely, the affinity for the tumor-associated isoforms by 1.2- and 2.0-fold. A similar kinetic trend was reported when the choro atom in 15a was placed instead (16a). Noteworthily, such a halogen showed a less significant reduction of the ligand affinity for hCAs I and II when compared to that of 15a with respect to the unsubstituted progenitor 14a. On the other hand, the inhibitory potency of the chloro-containing 16a on the tumor-associated hCAs was highly enhanced up to low nanomolar values (7.3 and 9.7 nM, respectively). It is of interest that the manipulation of our derivatives with halogens, although at a very simple level, had a great impact on the potency and selectivity of the ligands against the various isozymes considered. Again, the isosteric substitution of the oxygen in 15a and 16a with sulfur instead, as in 15b and 16b, showed significant K_I variations. As reported in Table 1, the inhibitory potency of 15b against hCAs I and XII was enhanced by 2.9- and 4.0-fold, respectively, when compared to its oxo progenitor 15a (K_Is for 15a = 272 and 289 nM for hCAs I and XII, respectively; K_Is for 15b = 95 and 72 nM for hCAs I and XII, respectively). On the contrary, the inhibitory potency of 15b against the remaining isoforms II and IX decreased by 3.5- and 1.7-fold, respectively. As regards 16b, a specific kinetic trend was reported for all the investigated hCAs, with all K_I values enhanced when compared to the progenitor 16a.

The introduction of the carboxamide at position 5 of the 6-phenyl-tetrahydropyrimidine ring as in 17a induced a significative and selective inhibition of the tumor-associated isoforms IX and XII (K_Is of 6.6 and 8.9 nM, respectively) over the ubiquitous hCAs I and II (K_Is of 217 and 70.5 nM, respectively). The isosteric manipulation performed on 17a to afford 17b maintained the kinetic trend and, specifically, spoiled the K_I values for hCAs I, II, and IX but resulted in being ineffective for the isoform XII.

Similar SARs were obtained for pyrimidintrione derivatives 20 and 21 and a preferential inhibition of hCAs II and IX was clearly observed in both cases. Methyl substitutions on the pyrimidine moiety in 20 greatly enhanced the binding affinity for hCA I (K_Is = 957 and 97 nM for 20 and 21, respectively), and slightly affected that for hCA II (K_Is = 29.1 and 17.3 nM for 20 and 21, respectively). Conversely, such a structural modification reduced the inhibitory potency for the tumor-associated IX and XII isoforms, with the latter being particularly affected (by 1.7-fold).

Among the series, 6-(chloromethyl)-tetrahydropyrimidine 13a and 2-aminopyrimidine 23 have structural peculiarities that set them apart. Besides such differences, both 13a and 23 shared similar kinetic preferentiality for hCAs IX and XII over the I and II isoforms. Among them, a slight selectivity was reported, with 13a being 2.4-fold more potent against hCA IX than against the isoform XII (K_Is = 3.5 and 8.5 nM, respectively), whereas 23 was 1.7-fold more potent against hCA XII than against the isoform IX (K_Is of 8.9 and 5.3 nM, respectively).

As for the CNS-associated hCA VII, isosteric substitution on 10a by the introduction of the sulfur atom was proved to be detrimental to the inhibitory potency (K_Is = 19.7 and 63.8 nM for 10a and 10b, respectively). The same in vitro kinetic trend was reported for 11a and 11b, although with moderate intensity (K_Is = 12.3 and 14.5 nM, respectively). Among the 5-ethyl ester-tetrahydropyrimidin-4-yl containing moiety series, the thiooxo derivative 12b was 1.7-fold more potent than its precursor 12a (K_Is = 15.5 and 25.7 nM for 12b and 12a, respectively). On the other hand, 6-phenyl derivative 14b was a 18.9-fold weaker inhibitor of hCA VII when compared to 14a, with K_Is equal to 191 and 10.1 nM, respectively. The halogen effect was clearly shown also for the hCA VII in vitro kinetics. For instance, the introduction of the fluorine and chlorine atoms in 14a to afford 15a and 16a, respectively, spoiled the inhibitory potency by 10.0- and 2.5-fold (K_Is = 102 and 25.0 nM for 15a and 16a), respectively. Quite interestingly, the sulfur isosteric manipulation on 14a and 15a heavily affected their K_I values, which were raised by 18.9- and 1.6-fold for 14b and 15b, respectively. A slight improvement (by 1.4-fold) of the binding affinity was observed for 16b compared to its precursor 16a (K_Is = 25.0 and 17.8 nM for 16a and 16b, respectively). Also, a notable increase in inhibitory potency on hCA VII was observed for thiooxo 17b when compared to its oxo progenitor (K_Is = 88.0 and 18.1 nM for 17a and 17b, respectively).

Among the pyrimidintriones 20 and 21, the methyl substitution produced a remarkable enhancement of the inhibitory potency up to 22.2-fold. Finally, 13a and 23 showed K_I values equal to 18.5 and 433 nM, respectively.

The selectivity index (SI) for each compound was calculated and a heat map is reported in ESI† (Fig. S1). In general, a good SI can be observed for the whole library of compounds over hCA I and hCA VII, while they were potent also against hCA II, apart from compound 14a, which preferentially inhibits the physiologically relevant isoforms I and II. Notably, compounds 10b and 17a were found to be the most selective in the series, with the latter showing a high selectivity for hCA IX (SI in the range of 10–100). Finally, compound 23 emerged as a selective inhibitor of tumor-associated isoforms, with a SI value >10.

Crystallographic data

We conducted an X-ray crystallographic investigation to decipher the binding mode of representative compounds of the series within the active site of hCAs considered in this study. Tetrahydropyrimidine 12a was the only one that afforded an adduct with the isoform I stable enough to be considered for our purposes.⁴² In addition, such an isoform is highly prevalent in red blood cells at a relevant concentration (i.e., up to 1 μM) and, thus, retains high value in pharmacokinetics. The electron density map revealed the presence of the inhibitor within the hCA I active site, clearly attributable to compound 12a (Fig. S2 in ESI†). This binding mode is facilitated by the interaction between the primary sulfonamide group in its anionic form and the protein Zn²⁺ ion (Fig. 2).

Fig. 2 hCA I in complex with 12a. residues involved in the binding of inhibitors are also shown; the grey sphere represents the zinc ion within the enzyme active site. Interactions between the inhibitor and enzyme residues are also shown: blue hydrophobic interactions; red hydrogen bonds; and green π–π stacking interactions.

The inhibitor also forms a robust hydrogen bond between one oxygen atom of the sulfonamide group and the amide nitrogen of Thr199. This coordination pattern is consistent with the typically reported primary sulfonamides.⁴² Furthermore, the inhibitor is firmly stabilized within the active site through several interactions. There is a T-shaped π-stacking interaction between the phenyl ring and His94 at the base of the cavity. Additionally, a hydrophobic interaction occurs with the side chain of Leu198 (Fig. 2). The inhibitor tail in 12a is engaged in hydrophobic interactions with Ala135 and forms two hydrogen bonds with the side chain of Gln92. Such a dual interaction with Gln92 is probably due to the presence of a keto-enolic equilibrium not clearly observable within the conducted experiments. In Table S1,† a summary of the data collected for cocrystal hCA I and compound 12a is reported.

Molecular docking simulations on the hCA XII isoform

The three-dimensional structure of hCA XII in complex with a benzenesulfonamide has been used to predict the putative binding mode of 12a by using Glide software (Maestro suite, Schrodinger). As a result, the best-scored binding pose of 12a and the co-crystallized ligand showed a similar location and orientation of their common benzenesulfonamide portion, which was predicted in the deprotonated form as the preferred tautomer. In addition to serving as a chelating group for the zinc ion, the sulfonamide group was involved in a network of hydrogen bonds (Fig. 3).

Fig. 3 Graphical representation of the best-scored pose of 12a (green carbon atoms and atom type notation) within the binding site of hCA XII (PDB ID: 6YH9). For the sake of clarity, only amino acids important for binding interactions are shown, in addition to his residues and zinc ion (blue sphere) of the catalytic machinery. Hydrogen bonds are represented by orange dotted lines.

In particular, the NH system is a hydrogen bond donor for the hydroxyl group of the Thr199 side chain, while the amide nitrogen atom served as an acceptor for the backbone NH group of Thr199, which in turn also interacted with one of the sulfone oxygen atoms. An additional hydrogen bond was found between sulfone oxygen and the backbone NH of Thr200. Moreover, the complex was further stabilized by a bifurcated hydrogen bond between the pyrimidone oxygen and the terminal ammonium group of Lys67 and with the NH₂ group of Gln92. Finally, the ethyl group of the ester side chain was accommodated within a hydrophobic cage delimited by the alkyl portions of Leu141, Leu198, and Ser135.

In vitro anti-proliferative activity

Representative compounds 13a, 17a–b, and 23 were selected for their potent inhibitory profile on cancer-associated isoforms hCAs IX and XII and their selectivity over the physiologically relevant I and II isoforms. Along with SLC-0111 (Fig. 1), the antiproliferative effect of the compounds was evaluated on a human triple-negative breast cancer cell line (MDA-MB-231, Fig. 4) and a human glioblastoma cell line (U87MG, Fig. 5) with a high basal expression of hCA IX.^22,43 Thus, the cells were treated with the compounds at different concentrations and their efficacy to inhibit the proliferation was assessed after 72 h of treatment in hypoxic conditions by cytofluorimetric cell count (Fig. 4 and 5). As already reported,^22,43 the reference compound SLC-0111 was effective in reducing the proliferation of MDA-MB-231 (IC₅₀ = 138 μM). Interestingly, 13a and 23 showed an enhanced antiproliferative effect, with IC₅₀ values of 44.4 and 38.8 μM, respectively. Otherwise, a lack of activity was observed for compounds 17a–b at 300 μM (Fig. 4).

Fig. 4 Cell proliferation of triple-negative breast cancer MDA-MB-231 cells treated for 72 h with 13a, 17a, b, 23, or SLC-0111. Cell count is referred to the untreated/control considered as 100%.

Fig. 5 Cell proliferation of U87MG glioblastoma cells treated for 72 h with 13a, 17a, b, 23, or SLC-0111. Cell count is referred to the untreated/control considered as 100%. Cell count is referred to the untreated/control considered as 100%.

Moreover, as shown in Fig. 5, SLC-0111 showed an IC₅₀ of 58 μM on U87MG cells, while 23 was the most effective (IC₅₀ = 34 μM) and an IC₅₀ equal to 100 μM was reported for 13a. Once again, 17a–b showed no relevant antiproliferative activity on the cell line (Fig. 5).

Experimental

Synthesis of the compounds

General chemistry. Anhydrous solvents and all reagents were purchased from Merck, VWR, and TCI. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere. Nuclear magnetic resonance (¹H and ¹³C NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d₆. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet. The assignment of exchangeable protons (NH) was confirmed by the addition of D₂O. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck silica gel 60 (230–400 mesh ASTM) as the stationary phase, and EtOAc and hexane were used as eluents. The solvents used in MS measurements were acetone, ACN (Chromasolv grade), purchased from Merck (Milan, Italy), and mQ water 18 MΩ, obtained from Millipore's Simplicity system (Milan, Italy). The mass spectra were obtained using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped with an electrospray source (ESI) operating in both positive and negative ions. Stock solutions of analytes were prepared in acetone at 1.0 mg mL⁻¹ and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of mQ H₂O/ACN 1/1 (v/v) up to a concentration of 1.0 μg mL⁻¹. The mass spectra of each analyte were acquired by introducing, via a syringe pump at 10 L min⁻¹, the working solution. Raw data were collected and processed using Varian Workstation version 6.8 software.

Synthetic procedures.
General procedure for the synthesis of DHPM-benzenesulfonamide 10–17a–b. A mixture of 4-formyl benzenesulfonamide (0.5 mmol), suitable β-dicarbonyl compounds (0.5 mmol), urea or thiourea (0.6 mmol), and NaHSO₄ (1.5 mmol) in hexane/ACN (2.5 : 0.5 mL) was refluxed for 6–24 h. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was cooled to r.t., and H₂O (5 mL) was added. The obtained precipitate was filtered and washed with cold water and Et₂O. The crude product was purified by column flash chromatography on silica gel using hexane/EtOAc 1 : 1 as the eluent.³⁸
4-(5-Acetyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-4-yl)benzenesulfonamide (10a). Yield 46%; yellow solid; mp: 237–238 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 2.19 (3H, s, –CH₃), 2.34 (3H, s, –CH₃), 5.36 (1H, d, J = 2.8 Hz, H–C₄), 7.35 (2H, s, –NH₂), 7.44 (2H, d, J = 8.4 Hz), 7.80 (2H, d, J = 8.4 Hz), 7.95 (1H, s, –NH), 9.31 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 20.1(CH₃), 31.6 (CH₃), 54.4 (CH), 110.6 (C), 127.1 (CH ×2), 128.0 (CH ×2), 144.1 (C), 149.1 (C), 149.8 (C), 153.1 (C=O), 195.1 (C=O) ppm; LC-MS (ESI) (m/z) 310.2 [M + H]⁺.
4-(5-Acetyl-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-4-yl)benzenesulfonamide (10b). Yield 32%; orange solid; mp: 218–219 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 2.24 (3H, s, –CH₃), 2.39 (3H, s, –CH₃), 5.39 (1H, d, J = 3.6 Hz, H–C4), 7.37 (2H, s, –NH₂), 7.43 (2H, d, J = 8.4 Hz), 7. 83 (2H, d, J = 8.4 Hz), 9.87 (1H, s, –NH), 10.42 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 19.4 (CH₃), 31.7 (CH₃), 54.4 (CH), 111.4 (C), 127.2 (CH ×2), 128.1 (CH ×2), 144.4 (C), 146.2 (C), 147.6 (C), 175.4 (C=S), 195.7 (C=O) ppm; LC-MS (ESI) (m/z): 326.2 [M + H]⁺.
4-(5-Benzoyl-2-oxo-6-phenyl-1,2,3,4-tetrahydropyrimidin-4-yl)benzenesulfonamide (11a). Yield 33%; off-white solid; mp: 267 °C (dec.); ¹H NMR (400 MHz, DMSO-d₆) δ: 5.43 (1H, d, J = 3.2 Hz, H–C4), 7.04 (2H, t, J = 7.6 Hz), 7.10 (2H, t, J = 7.6 Hz), 7.15–7.19 (2H, m), 7.24 (2H, d, J = 7.2 Hz), 7.29 (2H, d, J = 7.2 Hz), 7.36 (2H, s, –NH₂), 7.66 (2H, d, J = 8.0 Hz), 7.86 (2H, d, J = 8.0 Hz), 8.12 (1H, d, J = 3.2 Hz, –NH), 9.56 (1H, bs, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 56.2 (CH), 109.7 (C), 127.0 (CH ×2), 128.2 (CH ×2), 128.5 (CH ×2), 128.8 (CH ×2), 129.6 (CH ×2), 130.8 (CH ×2), 130.9 (CH), 131.7 (CH), 134.3 (C), 140.5 (C), 144.1 (C), 148.9 (C), 150.2 (C), 153.5 (C=O), 195.7 (C=O) ppm; LC-MS (ESI) (m/z): 434.2 [M + H]⁺.
4-(5-Benzoyl-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-4-yl)benzenesulfonamide (11b). Yield 75%; yellow solid; mp: 165–166 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 5.43 (1H, d, J = 2.8 Hz, H–C4), 7.05–7.21 (6H, m), 7.25 (2H, d, J = 7.2 Hz), 7.32 (2H, d, J = 7.6 Hz), 7.38 (2H, s, –NH₂), 7.65 (2H, d, J = 8.0 Hz), 7.89 (2H, d, J = 8.4 Hz), 10.01 (1H, s, –NH), 10.74 (1H, bs, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 56.0 (CH), 110.3 (C), 127.2 (CH ×2), 128.2 (CH ×2), 128.6 (CH ×2), 128.8 (CH ×2), 129.6 (CH ×2), 131.1 (CH ×2), 131.7 (C), 132.1 (CH), 133.3 (CH), 139.9 (C), 144.4 (C), 146.9 (C), 147.5 (C), 176.2 (C=S), 195.7 (C=O) ppm; LC-MS (ESI) (m/z): 450.2 [M + H]⁺.
Ethyl 6-methyl-2-oxo-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12a). Yield 65%; pale yellow solid; mp: 258–259 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.14 (3H, t, J = 6.8 Hz, CH₂–C̲H̲_3̲), 2.30 (3H, s, –CH₃), 4.02 (2H, q, J = 6.8 Hz, –CH₂), 5.24 (1H, s, H–C4), 7.36 (2H, s, –NH₂), 7.44 (2H, d, J = 7.2 Hz), 7.81 (2H, d, J = 7.2 Hz), 7.87 (1H, s, –NH), 9.34 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 15.1 (CH₃), 18.9 (CH₃), 54.8 (CH), 60.4 (CH₂), 99.6 (C), 127.0 (CH ×2), 127.9 (CH ×2), 144.1 (C), 149.5 (C), 150.0 (C), 153.00 (C=O), 166.2 (C=O) ppm; LC-MS (ESI) (m/z): 340.1 [M + H]⁺.
Ethyl 6-methyl-4-(4-sulfamoylphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12b). Yield 69%; white solid; mp: 224–225 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.12 (3H, t, J = 7.2 Hz, CH₂–C̲H̲_3̲), 2.32 (3H, s, CH₃), 4.03 (2H, q, J = 7.2 Hz), 5.25 (1H, s, H–C4), 7.34 (2H, s, –NH₂), 7.41 (2H, d, J = 8.4 Hz), 7.82 (2H, d, J = 8.4 Hz), 9.72 (1H, s, –NH), 10.43 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 15.1 (CH₃), 18.3 (CH₃), 54.8 (CH), 60.8 (CH₂), 101.1 (C), 127.2 (CH ×2), 128.1 (CH ×2), 144.4 (C), 146.7 (C), 148.0 (C), 166.0 (C=O), 175.4 (C=S) ppm; LC-MS (ESI) (m/z): 356.2 [M + H]⁺.
Ethyl 6-(chloromethyl)-2-oxo-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13a). Yield 72%; white solid; mp: 204–205 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.17 (3H, t, J = 7.2 Hz, –CH₃), 4.09 (2H, q, J = 7.2 Hz, –C̲H̲_2̲CH₃), 4.60 (1H, d, J = 10.8 Hz, –CH̲_a̲H_bCl), 4.83 (1H, d, J = 11.2 Hz, –CH_aH̲_b̲Cl), 5.30 (1H, d, J = 2.8 Hz, H–C4), 7.37 (2H, s, –NH₂), 7.46 (2H, d, J = 8.4 Hz), 7.83 (2H, d, J = 8.4 Hz), 7.98 (1H, bs, –NH), 9.63 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 15.0 (CH₃), 40.3 (CH₂), 54.7 (CH), 61.2 (CH₂), 102.1 (C), 127.1 (CH ×2), 127.9 (CH ×2), 144.4 (C), 147.7 (C), 148.6 (C), 152.9 (C=O), 165.1 (C=O) ppm; LC-MS (ESI) (m/z): 374.1 [M + H]⁺.
Ethyl 2-oxo-6-phenyl-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (14a). Yield 34%; yellow solid; mp: 236–237 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.75 (3H, t, J = 7.2 Hz, –CH₃), 3.75 (2H, q, J = 7.2 Hz, –CH₂), 5.33 (1H, d, J = 2.8 Hz, H–C4), 7.37 (2H, d, J = 7.6 Hz), 7.39 (2H, s, –NH₂), 7.41–7.47 (3H, m), 7.60 (2H, d, J = 8.4 Hz), 7.88 (2H, d, J = 8.0 Hz), 7.98 (1H, bs, –NH), 9.44 (1H, bs, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.4 (CH₃), 55.0 (CH), 60.3 (CH₂), 100.8 (C), 127.2 (CH ×2), 128.0 (CH ×2), 128.8 (CH ×2), 129.4 (CH ×2), 130.1 (CH), 136.0 (C), 144.2 (C), 149.2 (C), 150.6 (C), 153.0 (C=O), 166.1 (C=O) ppm; LC-MS (ESI) (m/z): 402.2 [M + H]⁺.
Ethyl 6-phenyl-4-(4-sulfamoylphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (14b). Yield 64%; beige solid; mp: 240–241 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.77 (3H, t, J = 7.2 Hz, –CH₃), 3.79 (2H, q, J = 7.2 Hz, –CH₂), 5.37 (1H, d, J = 3.6 Hz, H–C4), 7.37 (2H, d, J = 7.2 Hz), 7.40 (2H, s, –NH₂), 7.42–7.50 (3H, m), 7.59 (2H, d, J = 8.4 Hz), 7.90 (2H, d, J = 8.4 Hz), 9.88 (1H, s, –NH), 10.64 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.4 (CH₃), 54.9 (CH), 60.7 (CH₂), 102.3 (C), 127.3 (CH ×2), 128.1 (CH ×2), 128.8 (CH ×2), 129.8 (CH ×2), 130.3 (CH), 134.9 (C), 144.5 (C), 147.4 (C), 147.6 (C), 165.9 (C=O), 175.7 (C=S) ppm; LC-MS (ESI) (m/z): 418.2 [M + H]⁺.
Ethyl 6-(4-fluorophenyl)-2-oxo-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15a). Yield 73%; off-white solid; mp: 256–257 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.80 (3H, t, J = 6.4 Hz, –CH₃), 3.77 (2H, q, J = 6.4 Hz, –CH₂), 5.34 (1H, s, H–C4), 7.26 (2H, t, J = 8.0 Hz), 7.38 (2H, s, –NH₂), 7.40–7.47 (2H, m), 7.60 (2H, d, J = 7.2 Hz), 7.88 (2H, d, J = 8.0 Hz), 7.98 (1H, bs, –NH), 9.44 (1H, bs, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.5 (CH₃), 55.0 (CH), 60.4 (CH₂), 101.0 (C), 115.7 (CH ×2, ²J_C–F = 21.7 Hz), 127.2 (CH ×2), 128.0 (CH ×2), 131.9 (CH ×2, ³J_C–F = 8.4 Hz), 132.2 (C), 144.2 (C), 149.1 (C), 149.6 (C), 152.9 (C=O), 163.6 (C, ¹J_C–F = 244.2 Hz), 165.9 (C=O) ppm; LC-MS (ESI) (m/z): 418.2 [M–H]⁻.
Ethyl 6-(4-fluorophenyl)-4-(4-sulfamoylphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15b). Yield 33%; white solid; mp: 230–231 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.82 (3H, t, J = 6.8 Hz, –CH₃), 3.81 (2H, q, J = 6.8 Hz, –CH₂), 5.37 (1H, d, J = 3.6 Hz, H–C4), 7.27 (2H, t, J = 8.8 Hz), 7.40 (2H, s, –NH₂), 7.42–7.46 (2H, m), 7.58 (2H, d, J = 8.0 Hz), 7.89 (2H, d, J = 8.0 Hz), 9.88 (1H, s, –NH), 10.66 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.5 (CH₃), 54.9 (CH), 60.8 (CH₂), 102.5 (C), 115.7 (CH ×2, ²J_C–F = 21.7 Hz), 127.3 (CH ×2), 128.1 (CH ×2), 131.2 (C, ⁴J_C–F = 2.9 Hz), 132.2 (CH ×2, ³J_C–F = 8.3 Hz), 144.6 (C), 146.4 (C), 147.6 (C), 163.8 (C, ¹J_C–F = 245.1 Hz), 165.7 (C=O), 175.6 (C=S) ppm; LC-MS (ESI) (m/z): 436.2 [M + H]⁺.
Ethyl 6-(4-chlorophenyl)-2-oxo-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16a). Yield 70%; white solid; mp: 260–261 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.81 (3H, t, J = 7.2 Hz, –CH₃), 3.78 (2H, q, J = 7.2 Hz, –CH₂), 5.34 (1H, d, J = 2.4 Hz, H–C4), 7.39 (2H, s, –NH₂), 7.40 (2H, d, J = 8.8 Hz), 7.50 (2H, d, J = 8.0 Hz), 7.59 (2H, d, J = 8.0 Hz), 7.87 (2H, d, J = 7.6 Hz), 8.00 (1H, s, –NH), 8.49 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.5 (CH₃), 55.0 (CH), 60.4 (CH₂), 101.1 (C), 127.2 (CH ×2), 128.0 (CH ×2), 128.9 (CH ×2), 131.5 (CH ×2), 134.6 (C), 134.7 (C), 144.3 (C), 149.0 (C), 149.4 (C), 152.8 (C=O), 165.8 (C=O) ppm; LC-MS (ESI) (m/z): 436.2 [M + H]⁺.
Ethyl 6-(4-chlorophenyl)-4-(4-sulfamoylphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16b). Yield 57%; white solid; mp: 265–266 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.80 (3H, t, J = 6.8 Hz, –CH₃), 3.78 (2H, q, J = 7.2 Hz, –CH₂), 5.34 (1H, s, H–C4), 7.39–7.42 (4H, m (2H, –NH₂; 2H, ArH)), 7.50 (2H, d, J = 7.6 Hz), 7.59 (2H, d, J = 7.6 Hz), 7.87 (2H, d, J = 8.0 Hz), 8.01 (1H, s, –NH), 9.50 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 14.5 (CH₃), 55.0 (CH), 60.4 (CH₂), 101.2 (C), 127.2 (CH ×2), 128.0 (CH ×2), 128.9 (CH ×2), 131.4 (CH×2), 134.8 (C), 144.3 (C), 149.0 (C), 149.4 (C), 152.8 (C), 165.8 (C=O), 176.2 (C=S) ppm; LC-MS (ESI) (m/z): 451.1 [M–H]⁻.
2-Oxo-6-phenyl-4-(4-sulfamoylphenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (17a). Yield 75%; white solid; mp: 281–282 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 5.37 (1H, s, H–C4), 6.11 (1H, bs, –NH), 6.82 (1H, bs, –NH), 7.37 (2H, s, –SO₂NH₂), 7.45 (5H, s), 7.59 (2H, d, J = 8.4 Hz), 7.73 (1H, s, –NH), 7.85 (2H, d, J = 8.0 Hz), 8.89 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 56.1 (CH), 106.8 (C), 126.9 (CH ×2), 128.3 (CH ×2), 129.3 (CH ×2), 129.7 (CH ×2), 130.3 (CH), 135.1 (C), 141.4 (C), 144.1 (C), 148.8 (C), 153.7 (C=O), 168.7 (C=O) ppm; LC-MS (ESI) (m/z): 373.2 [M + H]⁺.
6-Phenyl-4-(4-sulfamoylphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (17b). Yield 82%; beige solid; mp: 238–240 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 5.37 (1H, d, J = 3.2 Hz, H–C4), 6.31 (1H, bs, –NH), 6.97 (1H, bs, –NH), 7.38 (2H, s, –SO₂NH₂), 7.46 (5H, s), 7.79 (2H, d, J = 8.4 Hz), 7.87 (2H, d, J = 8.4 Hz), 9.59 (1H, s, –NH), 10.17 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 55.9 (CH), 108.5 (C), 127.1 (CH ×2), 128.3 (CH ×2), 129.3 (CH ×2), 130.0 (CH ×2), 130.6 (CH), 134.0 (C), 139.1 (C), 144.4 (C), 147.4 (C), 168.3 (C=O), 175.7 (C=S) ppm; LC-MS (ESI) (m/z): 389.2 [M + H]⁺.

General procedure for the synthesis of pyrimidintrione-benzenesulfonamides 20 and 21. To an aqueous solution of NaOH (650.0 mg, 6.4 mmol, 3.0 mL), a solution of the suitable pyrimidine-2,4,6(1H,3H,5H)-trione 18–19 (5.0 mmol) in EtOH (5.0 mL) was added slowly at 0 °C. Then, 4-formyl benzenesulfonamide 1 (925.0 mg, 5.0 mmol) in EtOH (5.0 mL) was added dropwise and the mixture was stirred for 16 h at r.t. After completion, the formed precipitate was filtered under vacuum and washed with EtOH and H₂O.
4-((2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)benzenesulfonamide (20). Yield 59%; white solid; mp: >300 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.55 (2H, s, –NH₂), 7.88 (2H, d, J = 8.0 Hz), 8.07 (2H, d, J = 8.4 Hz), 8.34 (1H, s, CH), 11.34 (1H, s, –NH), 11.51 (1H, s, –NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 122.3 (CH), 126.0 (CH ×2), 133.2 (CH ×2), 137.3 (C), 146.8 (C), 151.3 (C=O), 153.5 (C), 162.3 (C=O), 164.0 (C=O) ppm; LC-MS (ESI) (m/z): 294.0 [M–H]⁻.
4-((1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)benzenesulfonamide (21). Yield 52%; pale yellow solid; mp: 218–219 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 3.20 (3H, s, –CH₃), 3.28 (3H, s, –CH₃), 7.55 (2H, s, –NH₂), 7.89 (2H, d, J = 8.4 Hz), 8.02 (2H, d, J = 8.4 Hz), 8.43 (1H, s, CH) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ: 29.1 (CH₃), 29.7 (CH₃), 122.1 (CH), 128.0 (CH ×2), 132.9 (CH ×2), 137.6 (C), 146.7 (C), 152.2 (C=O), 154.4 (C), 161.1 (C=O), 162.8 (C=O) ppm; LC-MS (ESI) (m/z): 322.0 [M–H]⁻.

In vitro carbonic anhydrase inhibition assay

An Applied Photophysics stopped-flow instrument was used to evaluate the ability of the test compounds to inhibit the CA-catalyzed CO₂ hydration.⁴¹ Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH 7.4) as a buffer, and 20 mM Na₂SO₄ (to maintain constant 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 assessment of the kinetic parameters and inhibition constants. Enzyme concentrations varied between 5 and 12 nM. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used to determine the initial velocity. The uncatalyzed rates were calculated in the same manner and subtracted from the total observed rates. Stock solutions of each inhibitor (0.1 mM) were prepared in distilled–deionized H₂O and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at r.t. prior to the assay, to allow for the formation of the E–I complex.^44,45 The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation as reported earlier⁴⁶ and represent the mean from at least three different determinations. hCAs I and II were purchased from Merck, while hCAs VII, IX, and XII are recombinant and obtained in-house.^47,48

Crystallography

Crystallization and X-ray data collection. The crystal of hCA I was obtained using the hanging drop vapor diffusion method using a 24 well Linbro plate. 2 μL of 10 mg mL⁻¹ solution of hCA I in Tris-HCl 20 mM pH 9.0 were mixed with 2 μL of a solution of 28–31% PEG4000, 0.2 M sodium acetate, and 0.1 M Tris pH 8.5–9.0 and were equilibrated against the same solution at 296 K. The complex was prepared by soaking the hCA I native crystals in the mother liquor solution containing 12a at a concentration of 10 mM for two days. All crystals were flash-frozen at 100 K using a solution obtained by adding 15% (v/v) glycerol to the mother liquor solution as a cryoprotectant. Data on the crystal of the complex was collected using synchrotron radiation at the XRD2 beamline at Elettra Synchrotron (Trieste, Italy) with a wavelength of 0.971 Å and a DECTRIS Pilatus 6M detector. Data were integrated and scaled using the program XDS.⁴⁹ Data processing statistics are shown in ESI.†

Structure determination. The crystal structure of hCA I (PDB ID: 1JV0) without solvent molecules and other heteroatoms was used to obtain initial phases using Refmac5.⁵⁰ 5% of the unique reflections were selected randomly and excluded from the refinement data set for Rfree calculations. The initial |Fo − Fc| difference electron density maps unambiguously showed the inhibitor molecules. Refinements proceeded using normal protocols of positional, isotropic atomic displacement parameters alternating with the manual building of the models using COOT.⁵¹ The quality of the final models was assessed with COOT and RAMPAGE.⁵² Crystal parameters and refinement data are summarized in Supplementary Information. Atomic coordinates were deposited in the Protein Data Bank (PDB ID: 8QGV). Graphical representations were generated with UCSF Chimera.⁵³

Molecular modelling. The three-dimensional structure of the complex between the human carbonic anhydrase XII and a benzenesulfonamide derivative (PDB ID: 6YH9, 1.55 Å resolution)⁵⁴ was used to predict the putative binding mode of the pyrimidinone analogues. The complex was submitted to the Protein Preparation Wizard of the Maestro suite (release 2020-3, Schrodinger LLC, 2020) to assign bond orders, refine hydrogen position, and apply a restrained minimization of the resulting structure by using the OPLS3e force field. The structure of the pyrimidinones was built by the 3D Builder module of Maestro and submitted to the LigPrep routine to generate possible tautomers at pH 7.0 ± 1 and metal binding states using Epik software.

For docking calculations, the receptor grid generation routine was applied to specify the location and the size of the grid where the ligands will be docked in suitable poses and orientations. The centroid of the co-crystallized ligand was used to define the grid center, while the overall grid size was based on a ligand diameter midpoint box of 20 × 20 × 20 Å. The hydroxyl groups of Ser, Thr, and Tyr residues within the grid were allowed to rotate during simulations. Flexible and standard-precision docking calculations were performed with post-docking minimization of the resulting complexes. The final docking poses were prioritized based on the corresponding docking score.

Cell culture and proliferation assays

Human MDA-MB-231 cells (ATCC HTB-26, triple-negative breast cancer) and human U87MG (ATCC HTB-14, glioblastoma multiforme) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Lonza), 1% penicillin/streptomycin (Lonza), and 1% glutamine (Lonza). Cells were kept at the low passage, returning to their original frozen stocks every 3–4 months. Hypoxic culture conditions were obtained in the presence of 1% O₂ and 5% CO₂. For cell proliferation, 10.000 cells per well were plated in a 48-well plate and treated in 1% FBS with increasing concentrations of 13a, 17a, 17b, 23, or SLC-0111. After 72 h of incubation at 37 °C with 1% O₂ and 5% CO₂, cells were trypsinized and cell counting was performed with a MACSQuant® Analyzer (Miltenyi Biotec).

Interference compound assessment

The behavior of final compounds 10–17a–b, 20, 21, and 23 as pan-assay interference compounds (PAINS)⁵⁵ was evaluated through SwissADME.^56,57 JChem for Office (21.15.704, 2023) by ChemAxon⁵⁸ was used for structure management, SMILES generation, and file conversion. DHPMs 10–17a–b and 23 resulted in no PAIN alert, while the software reported an alert for 20 and 21 due to the presence of the barbituric scaffold.

Discussion and conclusion

A series of 15 Biginelli's products (10–17a–b, Table 1) decorated with the benzenesulfonamide group, a known CA inhibitor chemotype, along with 2 barbituric compounds (20–21, Table 1) were designed and synthesized to better explore the SARs. The compounds' inhibitory activity towards hCAs IX and XII, along with their selectivity over the physiologically relevant hCAs I and II, and the isoform VII was assessed, revealing a low-nanomolar potency and promising isoform selectivity for some compounds. Crystallographic experiments were conducted, allowing the obtainment of the hCA I–12a complex with a high resolution (1.84 Å) and a suboptimal Rfree value (0.24). The X-ray structure of the complex with hCA I (PDB ID: 8QGV) helped to gain a fundamental insight into the key interaction networks governing enzyme inhibition. However, attempts to cocrystallize the inhibitor with tumor-associated isoforms failed and docking simulations were performed, revealing the binding pose of 12a in the catalytic pocket of hCA XII.

Furthermore, derivative 17a, bearing an amide function in position 5 of the pyrimidindione ring, was one of the most potent and selective compounds against the tumor-associated CAs (in terms of K_I and SI values), and, thus, was used as a hit compound for a preliminary screening campaign of in-house libraries of derivatives (not shown). 2-Aminopyrimidine derivative 23 showed a sufficient structural similarity (AP Tanimoto coefficient ∼0.5, calculated by means of the web tool ChemMine),^59,60 to be worthy of investigation. Surprisingly, enzymatic assays of these compounds revealed an unexpectedly high nanomolar potency and selectivity for hCAs IX and XII (Table 1 and Fig. S1†). Thus, representative compounds were selected in the Biginelli series, including 17a, and pyrimidine 23, for an in vitro investigation of their putative antiproliferative effect on MDA-MB-231 and U87MG cells. These cell lines have been selected due to the recent evidence of hCA IX overexpression in breast cancer,⁴³ a disease affecting millions of people annually – with 2.3 million women receiving the diagnosis and more than 600 000 deaths in 2020,⁶¹ and glioblastoma.²² Compounds 13a and 23 were effective in reducing the proliferation of the breast cancer cells, with lower IC₅₀ values than the reference CA inhibitor SLC-0111, and of glioblastoma cells. These results confirm the inhibition of CAs IX and XII as a promising mode of action for anticancer agent development. However, a discrepancy in the activity data was noticed for compound 17a, probably due to the permeability issues.

Remarkably, the new 2-aminopyrimidine scaffold contained in compound 23 emerged from a preliminary screening based on the chemical similarity of in-house libraries. Although a non-encouraging premise (non-optimal Tanimoto coefficient), 23 displayed high potency and selectivity as a hCA IX and XII inhibitor and a notable antiproliferative effect on breast cancer cells, thereby suggesting it as a valuable starting point for the development of a new derivative library.

Author contributions

The manuscript was written through the contributions of all authors. All authors approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Hakan Aslan acknowledges the financial support from The Scientific and Technological Research Council of Turkey (TUBITAK) BIDEB-2219 Postdoctoral Research Program (1059B192101059).

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Footnote

† Electronic supplementary information (ESI) available: Chemistry, heat map for selectivity indexes, electron density maps of inhibitor 12a, chemistry – experimental, NMR spectra of final compounds. See DOI: https://doi.org/10.1039/d4md00101j

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