Uracil as a Zn-Binding Bioisostere of the Allergic Benzenesulfonamide in the Design of Quinoline–Uracil Hybrids as Anticancer Carbonic Anhydrase Inhibitors

A series of quinoline–uracil hybrids (10a–l) has been rationalized and synthesized. The inhibitory activity against hCA isoforms I, II, IX, and XII was explored. Compounds 10a–l demonstrated powerful inhibitory activity against all tested hCA isoforms. Compound 10h displayed the best selectivity profile with good activity. Compound 10d displayed the best activity profile with minimal selectivity. Compound 10l emerged as the best congener considering both activity (IC50 = 140 and 190 nM for hCA IX and hCA XII, respectively) and selectivity (S.I. = 13.20 and 9.75 for II/IX, and II/XII, respectively). The most active hybrids were assayed for antiproliferative and pro-apoptotic activities against MCF-7 and A549. In silico studies, molecular docking, physicochemical parameters, and ADMET analysis were performed to explain the acquired CA inhibitory action of all hybrids. A study of the structure–activity relationship revealed that bulky substituents at uracil N-1 were unfavored for activity while substituted quinoline and thiouracil were effective for selectivity.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that are distributed extensively throughout living organisms [1]. CAs are subclassified to eight gene families (α, β, γ, δ, ζ, η, θ, and ι) [2]. As these enzymes are ubiquitously represented in the body, they are major participants in reaction catalysis and physiological processes. In addition to their physiological impacts, the modulation of the natural function or upregulation of these enzymes demonstrate immense benefits for controlling many pathological conditions [3]. The α-CA enzymes found in mammals are divided into four subgroups consisting of several isoforms. The CA I and CA II isoforms belong to the cytosolic subgroup of α-CA, while CA IX and CA XII isoforms are part of the membraneassociated α-CAs subgroup [4]. For instance, sulfonamides were determined as potent inhibitors of α-CAs and can be used to exploit many clinical applications such as diuresis, antiglaucoma, anticonvulsant, antiobesity, analgesic, and anticancer activities [5].
Cancer one of the leading mortality-causing diseases, competing with cardiovascular diseases for first place. It is also one of the overwhelming barriers to high life expectancy owing to its social and economic consequences [6]. In 2020, 19.3 million people were diagnosed with cancer, with 10.0 million deaths reported. This number is expected to reach 28.4 million in 2040 [7]. Many molecular mechanisms and biological targets are connected to the underlying mechanisms of cancer. Of these, human carbonic anhydrases (hCA) IX and XII have been recognized as tumor-associated proteins in hypoxic and other solid tumors where they actively contribute to the permanence and metastasis of the necrotic cell [8]. Notably, a great number of scientists have emphasized that both are biomarkers and therapeutic targets for various cancer types. Accordingly, dual targeting, i.e., inhibition, of the two latter isozymes represent a remarkable challenge for the development of novel anticancer drugs [9][10][11][12].
Following the successful impact of its modulation, the search for further pharmacophor es-other than sulfonamides-that can fit/block the active site of CAs has become an interesting topic in medicinal chemistry research [13][14][15]. Afterwards, many chemical moieties have been revealed as isosteres for sulfonamides, such as hydroxybenzene, fullerenes, benzopyrone, thiobenzopyrone, and boronic acids [3]. These new effective chemical pharmacophores have fostered the ability to design promising pharmacologically active candidates that circumvent sulfonamide side effects. In addition, they support further understanding of the underlying mechanisms of CAs and can be utilized for the rational drug design of novel candidates as CAs modulators.
It is important to indicate that CAs are not the sole Zn-containing enzymes that extensively affect cancer physiology [16]. Histone deacetylases (HDACs) are Zn-containing enzymes that play a crucial role in anticancer therapy [17]. Fortunately, the zinc-binding groups (ZBGs) of HDAC inhibitors are different from those reported for CAs, as per the recent reviews [18,19]. The ZBGs of HDACIs are classified into classical and nonclassical groups. The classical ZBGs, such as hydroxamic acid [20] and benzamide [21], are characterized by potent activity, selectivity, toxicity, and in vivo instability [22]. Carboxylic acid [23] and thiol [24] groups were also used as ZBGs in HDACI design. The nonclassical type include imidazole-thione [25], tropolone derivatives [26], 3-hydroxypyridin-2-thione (3-HPT) [27], chelidamic derivatives [28], benzoylhydrazide [29], trifluoromethyloxadiazolyl (TFMO) [30], 2-(oxazol-2-yl)phenol [31], hydroxypyrimidines [32], and β-hydroxymethyl chalcone [33] (Figure 1). Cancer one of the leading mortality-causing diseases, competing with cardiovascular diseases for first place. It is also one of the overwhelming barriers to high life expectancy owing to its social and economic consequences [6]. In 2020, 19.3 million people were diagnosed with cancer, with 10.0 million deaths reported. This number is expected to reach 28.4 million in 2040 [7]. Many molecular mechanisms and biological targets are connected to the underlying mechanisms of cancer. Of these, human carbonic anhydrases (hCA) IX and XII have been recognized as tumor-associated proteins in hypoxic and other solid tumors where they actively contribute to the permanence and metastasis of the necrotic cell [8]. Notably, a great number of scientists have emphasized that both are biomarkers and therapeutic targets for various cancer types. Accordingly, dual targeting, i.e., inhibition, of the two latter isozymes represent a remarkable challenge for the development of novel anticancer drugs [9][10][11][12].
Following the successful impact of its modulation, the search for further pharmacophores-other than sulfonamides-that can fit/block the active site of CAs has become an interesting topic in medicinal chemistry research [13][14][15]. Afterwards, many chemical moieties have been revealed as isosteres for sulfonamides, such as hydroxybenzene, fullerenes, benzopyrone, thiobenzopyrone, and boronic acids [3]. These new effective chemical pharmacophores have fostered the ability to design promising pharmacologically active candidates that circumvent sulfonamide side effects. In addition, they support further understanding of the underlying mechanisms of CAs and can be utilized for the rational drug design of novel candidates as CAs modulators.
In all the investigated compounds, the quinoline backbone was important for the biological activity; however, the zinc-binding group (primary benzenesulfonamide) is the Primary benzenesulfonamide derivatives are well known for their CA inhibition, which confers several biological activities, especially anticancer activity. In this regard, quinoline-based sulfonamides have been recently reported with impressive CA inhibition, where compound I displayed inhibitory activity against hCA I and II isoforms with Ki ranges of 0.96-9.09 µM and 83.3-3.59 µM for hCA I and II, respectively [53]. Compound II displayed potent inhibitory activity against the well-known cancer-related isozymes hCA IX and XII with Ki values of 0.019 µM and 0.009 µM, respectively [66].
In all the investigated compounds, the quinoline backbone was important for the biological activity; however, the zinc-binding group (primary benzenesulfonamide) is the essential moiety [53,[67][68][69][70]. Despite its key impact, the allergic response to sulfonamide drugs is a very unfavorable reaction [71,72]. Thus, the search for an alternative bioisostere is of much interest in the field of medicinal chemistry. Notably, uracil compounds III and IV are non-sulfonamide derivatives that show promising CA inhibitory activity and were confirmed as leads for generating potent CAIs, triggering further isoforms [73]. From the perspective of the structure-activity relationship, the uracil moiety with C=O, N, and NH 2 represents an active pharmacophore, rich with binding groups that can substitute the sulfonamide interactions in the receptor ( Figure 4).
represents an active pharmacophore, rich with binding groups that can substitute the sulfonamide interactions in the receptor ( Figure 4).
In light of the abovementioned findings, and by applying the tactic of pharmacophoric hybridization, a novel series of quinoline-based uracil synthetic molecules has been rationalized, synthesized, and tested for their CA inhibitory and anticancer activities. The utilized uracils with diverse functionality were similar to the primary benzenesulfonamide moiety regarding the two sites for both H-bond donors and H-bond acceptors. This rationale has promoted the exploration of such hybrids. These hybrid structures have been lined chemically via an imine bond (Schiff's base), which has been carefully selected as an anchor based on the reported CA inhibitory activity of its derivatives (compound V) [74] (Figure 4). An additional notable feature of our novel hybrids is the synthesis of quinoline-based thiouracil to realize the power of sulfur-based compounds as a zinc-binding group [75]. Docking and in silico studies have been performed to determine the CA inhibitory profile for the targeted quinolones.

Chemistry
The utilized protocol of our designed targets 10a-l consists of two key structural features; first, the quinoline-3-carbaldehyde moiety (Scheme 1), and second, the 5,6-diaminouracil/thiouracil pharmacophores (Scheme 2). The straightforward synthesis leading to 2- In light of the abovementioned findings, and by applying the tactic of pharmacophoric hybridization, a novel series of quinoline-based uracil synthetic molecules has been rationalized, synthesized, and tested for their CA inhibitory and anticancer activities. The utilized uracils with diverse functionality were similar to the primary benzenesulfonamide moiety regarding the two sites for both H-bond donors and H-bond acceptors. This rationale has promoted the exploration of such hybrids. These hybrid structures have been lined chemically via an imine bond (Schiff's base), which has been carefully selected as an anchor based on the reported CA inhibitory activity of its derivatives (compound V) [74] (Figure 4). An additional notable feature of our novel hybrids is the synthesis of quinoline-based thiouracil to realize the power of sulfur-based compounds as a zinc-binding group [75]. Docking and in silico studies have been performed to determine the CA inhibitory profile for the targeted quinolones.

Chemistry
The utilized protocol of our designed targets 10a-l consists of two key structural features; first, the quinoline-3-carbaldehyde moiety (Scheme 1), and second, the 5,6diaminouracil/thiouracil pharmacophores (Scheme 2). The straightforward synthesis leading to 2-quinolone-3-carbaldehyde derivatives (4a-c), which has benefit of concise- ness, is shown in Scheme 1. The acetylation of aniline derivatives 1a-c through glacial acetic acid/acetic anhydride at a temperature of 0 • C furnished the corresponding anilides (2a-c) [76,77]. These compounds underwent the Vilsmeier-Haack reaction to give the 2-chloroquinoline aldehyde derivatives (3a-c) [78,79]. The oxidation of the chlorine group at position 2 into a ketone has been successfully performed under reflux in acidic conditions (acetic acid) to deliver the desired synthons (4a-c). This reaction was initiated by oxidation with acetic acid, then completed by the departure of chlorine as a good leaving group to produce the favored product. The reaction appeared to be facilitated through the electron withdrawing effect of nitrogenous heteroatom [78,79]. quinolone-3-carbaldehyde derivatives (4a-c), which has benefit of conciseness, is shown in Scheme 1. The acetylation of aniline derivatives 1a-c through glacial acetic acid/acetic anhydride at a temperature of 0 °C furnished the corresponding anilides (2a-c) [76,77]. These compounds underwent the Vilsmeier-Haack reaction to give the 2-chloroquinoline aldehyde derivatives (3a-c) [78,79]. The oxidation of the chlorine group at position 2 into a ketone has been successfully performed under reflux in acidic conditions (acetic acid) to deliver the desired synthons (4a-c). This reaction was initiated by oxidation with acetic acid, then completed by the departure of chlorine as a good leaving group to produce the favored product. The reaction appeared to be facilitated through the electron withdrawing effect of nitrogenous heteroatom [78,79]. As shown in Scheme 2, the variable 5,6-diaminouracil/thiouracil derivatives 9a-e were prepared via consecutive cyclization of N-alkylurea/thiourea with ethyl cyanoacetate in the presence of sodium ethoxide, which initially gave 6-aminouracil/thiouracils 7ae. This was readily followed by conversion via a nitrosation process using nitrous acid and then reduction of nitroso uracil/thiouracils 8a-e via the reducing agent ammonium sulfide [80,81]. quinolone-3-carbaldehyde derivatives (4a-c), which has benefit of conciseness, is shown in Scheme 1. The acetylation of aniline derivatives 1a-c through glacial acetic acid/acetic anhydride at a temperature of 0 °C furnished the corresponding anilides (2a-c) [76,77]. These compounds underwent the Vilsmeier-Haack reaction to give the 2-chloroquinoline aldehyde derivatives (3a-c) [78,79]. The oxidation of the chlorine group at position 2 into a ketone has been successfully performed under reflux in acidic conditions (acetic acid) to deliver the desired synthons (4a-c). This reaction was initiated by oxidation with acetic acid, then completed by the departure of chlorine as a good leaving group to produce the favored product. The reaction appeared to be facilitated through the electron withdrawing effect of nitrogenous heteroatom [78,79]. As shown in Scheme 2, the variable 5,6-diaminouracil/thiouracil derivatives 9a-e were prepared via consecutive cyclization of N-alkylurea/thiourea with ethyl cyanoacetate in the presence of sodium ethoxide, which initially gave 6-aminouracil/thiouracils 7ae. This was readily followed by conversion via a nitrosation process using nitrous acid and then reduction of nitroso uracil/thiouracils 8a-e via the reducing agent ammonium sulfide [80,81]. As shown in Scheme 2, the variable 5,6-diaminouracil/thiouracil derivatives 9a-e were prepared via consecutive cyclization of N-alkylurea/thiourea with ethyl cyanoacetate in the presence of sodium ethoxide, which initially gave 6-aminouracil/thiouracils 7a-e. This was readily followed by conversion via a nitrosation process using nitrous acid and then reduction of nitroso uracil/thiouracils 8a-e via the reducing agent ammonium sulfide [80,81]. Condensation of diamino-uracil/thiouracils 9a-e with the appropriate quinoline aldehydes 4a-c under refluxing condition in ethanol for 1 h furnished the anticipated quinolinebased uracils/thiouracils 10a-l (Scheme 3). The structures of these novel compounds were also characterized by using thin-layer chromatography and melting point methods. Our novel later hybrids were determined using 1 H NMR, 13 C NMR, elemental, and mass spectroscopic techniques. The 1 H NMR spectra showed characteristic imino protons (N=CH) of Schiff bases appearing in the range from 8.45-9.91 ppm as sharp singlets. The 13 C NMR spectra further supported the assigned structures. The 13 C NMR shift of N=CH carbon atoms appeared in the range from 161.32-164.41 ppm as a singlet signal. Condensation of diamino-uracil/thiouracils 9a-e with the appropriate quinoline aldehydes 4a-c under refluxing condition in ethanol for 1 h furnished the anticipated quinoline-based uracils/thiouracils 10a-l (Scheme 3). The structures of these novel compounds were also characterized by using thin-layer chromatography and melting point methods. Our novel later hybrids were determined using 1 H NMR, 13 C NMR, elemental, and mass spectroscopic techniques. The 1 H NMR spectra showed characteristic imino protons (N=CH) of Schiff bases appearing in the range from 8.45-9.91 ppm as sharp singlets. The 13 C NMR spectra further supported the assigned structures. The 13 C NMR shift of N=CH carbon atoms appeared in the range from 161.32-164.41 ppm as a singlet signal.

Carbonic Anhydrase Inhibition
Activities of all CA isoenzymes were estimated following the previously described colorimetric method of Verpoorte et al. (1967) [82,83] using BioVision Carbonic Anhydrase (CA) Inhibitor Screening Kit (Catalog # K473-100). As per Table 1, all candidates 10a-l showed variable inhibitory activities against the tested CA isoforms. For hCAI, all the synthesized candidates 10a-l demonstrated inhibitory activities. Compounds 10a, 10d, 10f, 10j, and 10l exhibited inhibitory concentrations in the nanomolar range (230-970 nM), whereas the other compounds showed activity in the micromolar range (Table 1 and Figure 5). Compounds 10d, 10f, and 10l displayed higher inhibitory activity (IC50 = 230, 330, and 250 nM, respectively) than the reference standard acetazolamide (AAZ, IC50 = 760 nM). It is shown that the placement of small lipophilic methyl groups at N-1 uracil (where X = O), i.e., compound 10d, was preferred to X = S and the bulkier ethyl and benzyl groups.
In the case of the physiologically dominant hCA II, candidates 10a, 10c, 10d, and 10f showed potent inhibitory activity in the nanomolar range (IC50 = 520, 660, 260, and 790 nM, respectively, Table 1 and Figure 5). Similarly, compound 10d was found to be the most potent hCA II inhibitor with a lower inhibitory concentration (IC50 = 260 nM) than the reference standard acetazolamide (AAZ, IC50 390 nM). This highlights the effect of bulkiness on the interaction with the hCA II binding site. Fortunately, tumor-linked hCA IX was efficiently inhibited by most of our candidates in the nanomolar range, while three candidates (10e, 10g, and 10k) showed inhibition in the low micromolar range (IC50 = 1.37,

Carbonic Anhydrase Inhibition
Activities of all CA isoenzymes were estimated following the previously described colorimetric method of Verpoorte et al. (1967) [82,83] using BioVision Carbonic Anhydrase (CA) Inhibitor Screening Kit (Catalog # K473-100). As per Table 1, all candidates 10a-l showed variable inhibitory activities against the tested CA isoforms. For hCAI, all the synthesized candidates 10a-l demonstrated inhibitory activities. Compounds 10a, 10d, 10f, 10j, and 10l exhibited inhibitory concentrations in the nanomolar range (230-970 nM), whereas the other compounds showed activity in the micromolar range (Table 1 and Figure 5). Compounds 10d, 10f, and 10l displayed higher inhibitory activity (IC 50 = 230, 330, and 250 nM, respectively) than the reference standard acetazolamide (AAZ, IC 50 = 760 nM). It is shown that the placement of small lipophilic methyl groups at N-1 uracil (where X = O), i.e., compound 10d, was preferred to X = S and the bulkier ethyl and benzyl groups.

Antiproliferative Activity
Based on their activities and selectivity against tumor-linked CA isoforms, compounds 10d and 10l were selected for further screening against the breast cancer cell line (MCF-7) (HTB-22 from ATCC, Manassas, VA, USA) and lung cancer cell line (A549) (CCL-185 from ATCC, Manassas, VA, USA) under hypoxic conditions to evaluate their in vitro antiproliferative activity using the MTT assay protocol [84] (Table 3). Both compounds showed activity against the tested cell lines. However, compound 10d had more potent activity than 10l against the MCF7 breast cancer cell line with IC50 = 2.87 ± 0.05 compared with compound 10l IC50 value of 4.08 ± 0.08 µM. For the reference standard staurosporine, the IC50 was 6.92 ± 0.18 µM. Despite their superior activity over staurosporine against

Assessment of Apoptotic Marker Levels
The two cytoplasmic proteins, B-cell lymphoma protein 2 (Bcl-2) and its associated X protein (Bax), are essential for apoptosis in normal cells. Bax is a promoter and Bcl-2 is an

Antiproliferative Activity
Based on their activities and selectivity against tumor-linked CA isoforms, compounds 10d and 10l were selected for further screening against the breast cancer cell line (MCF-7) (HTB-22 from ATCC, Manassas, VA, USA) and lung cancer cell line (A549) (CCL-185 from ATCC, Manassas, VA, USA) under hypoxic conditions to evaluate their in vitro antiproliferative activity using the MTT assay protocol [84] (Table 3). Both compounds showed activity against the tested cell lines. However, compound 10d had more potent activity than 10l against the MCF7 breast cancer cell line with IC50 = 2.87 ± 0.05 compared with compound 10l IC50 value of 4.08 ± 0.08 µM. For the reference standard staurosporine, the IC50 was 6.92 ± 0.18 µM. Despite their superior activity over staurosporine against breast cancer cell line, compounds 10d and 10l had lower activity than staurosporine towards the lung cancer cell line (Figure 7).

Assessment of Apoptotic Marker Levels
The two cytoplasmic proteins, B-cell lymphoma protein 2 (Bcl-2) and its associated X protein (Bax), are essential for apoptosis in normal cells. Bax is a promoter and Bcl-2 is an inhibitor of apoptosis [85]. Thus, the effect of 10d and 10l on apoptotic markers Bax and Bcl-2 in a breast cancer cell line (MCF-7) and a lung cancer cell line (A549) has been estimated. Treatment of MCF-7 and A549 cell lines with compounds 10d and 10l resulted in the upregulation of Bax levels by nearly six-fold relative to the control while the expression of Bcl-2 levels was downregulated in comparison with the control (Table 3).

Physicochemical and Pharmacokinetic Parameters
The SWISSADME server [86] was utilized to assess the ability of the investigated compounds (10a-l) to act as drugs via estimating the physicochemical and pharmacokinetic properties. No compounds were expected to cross the BBB, inhibit cytochrome enzymes, violate the Lipinski's rule, or give PAINS alerts. In addition, all compounds (except 10g, 10h, and 10l) demonstrated high GIT absorption. The investigated compounds showed good synthetic accessibility and bioavailability scores (Table 4). Moreover, no compounds inhibited cytochrome P450 enzymes, indicating there was no expected pharmacokineticrelated drug-drug interactions.

Molecular Docking Study
Molecular docking studies were performed to investigate the interactions of the target compounds 10a-10l with the binding site of the human carbonic anhydrases IX using Discovery Studio. hCA IX is considered promising targets for cancer treatment and their inhibition can reduce the growth of primary tumors and metastases. For the CA IX isoform, the PDB file 5FL4 containing hCA IX co-crystallized with 5-(1-naphthalen-1-yl-1,2,3-triazol-4-yl)thiophene-2 sulfonamide was obtained from the Protein Data Bank [87]. Table 4. Predicted parameters of compounds 10a-l using SWISSADME server. The protein structure was prepared by 3D protonation and the water molecule and ligand that were not implicated in the active site were removed. The active site then generated with the default protocol [88]. The 2D and 3D interaction diagrams for the ligand and compounds 10d, 10f, and 10l are shown in Figure 8. Analyzing the ligand-protein interactions can help better understand the selectivity of the compound for hCA IX with respect to the other hCA isoforms.
The co-crystallized ligand forms two hydrogen bonds with Thr 200 and Leu199; it was found to chelate the zinc ion through the sulfonamide group.

SAR Study
Uracil is well documented as a metal-binding pharmacophore [89][90][91] with particular emphasis on its Zn +2 -binding abilities [92,93]. In the present work, uracil has been established as a bioisostere of the zinc-binding moiety benzenesulfonamide in our synthesized carbonic anhydrase inhibitors 10a-l. The uracil Zn-binding ability has been proven using modeling studies and realized by the biological screening against CA isoforms (I, II, IX, XII) and cancer cell lines.
The SAR of the synthesized candidates can be summarized as follows (Figure 9): -Both uracil and thiouracil had CA inhibitory activity. -Substitution on uracil N-1 with a bulky group (benzyl 10b and 10i) decreases activity. -Substitution on the quinoline ring has tolerable activity, but greatly improves the selectivity, particularly when in combination with thiouracil (10l).  modeling studies and realized by the biological screening against CA isoforms (I, II, IX, XII) and cancer cell lines. The SAR of the synthesized candidates can be summarized as follows (Figure 9): -Both uracil and thiouracil had CA inhibitory activity. -Substitution on uracil N-1 with a bulky group (benzyl 10b and 10i) decreases activity. -Substitution on the quinoline ring has tolerable activity, but greatly improves the selectivity, particularly when in combination with thiouracil (10l).

Chemistry
Melting points were determined with a Gallenkamp (London, UK) melting point apparatus and were uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Varian Gemini-400 (400MHz, Foster City, Calif., USA) spectrometer using chloroform (CDCl3), dimethylsulphoxide and/or (DMSO/D2O) as solvents and tetramethylsilane (TMS) as an internal standard (chemical shift in δ, ppm). 1 H NMR data were recorded as follows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), relative integral], where multiplicity is defined as s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet, or combinations of the above. High-resolution measurements were conducted on a time-offlight instrument. All the results of elemental analyses corresponded to the calculated values within experimental error. The reaction progress was observed by thin-layer chromatography (TLC) using TLC sheets precoated with ultraviolet (UV) fluorescent silica gel (Merck 60F254) and spots were visualized by irradiation with UV light (254 nm) or iodine vapors. All starting materials and reagents were generally commercially available and purchased from Sigma-Aldrich or Lancaster Synthesis Corporation (Lancaster, UK). Compounds 4a-c and 9a-e were prepared according to the reported method [80,81,[94][95][96].

Chemistry
Melting points were determined with a Gallenkamp (London, UK) melting point apparatus and were uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Varian Gemini-400 (400MHz, Foster City, Calif., USA) spectrometer using chloroform (CDCl 3 ) , dimethylsulphoxide and/or (DMSO/D 2 O) as solvents and tetramethylsilane (TMS) as an internal standard (chemical shift in δ, ppm). 1 H NMR data were recorded as follows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), relative integral], where multiplicity is defined as s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet, or combinations of the above. High-resolution measurements were conducted on a time-of-flight instrument. All the results of elemental analyses corresponded to the calculated values within experimental error. The reaction progress was observed by thin-layer chromatography (TLC) using TLC sheets precoated with ultraviolet (UV) fluorescent silica gel (Merck 60F254) and spots were visualized by irradiation with UV light (254 nm) or iodine vapors. All starting materials and reagents were generally commercially available and purchased from Sigma-Aldrich or Lancaster Synthesis Corporation (Lancaster, UK). Compounds 4a-c and 9a-e were prepared according to the reported method [80,81,[94][95][96].

Molecular Modeling Study
The molecular modeling studies were fulfilled by the Molecular Operating Environment software (Discovery Studio). The crystal structure for hCA IX co-crystallized with 5-(1-naphthalen-1-yl-1,2,3-triazol-4-yl) thiophene-2-sulfonamide was downloaded from the Protein Data Bank (PDB ID: 5FL4) [87]. The protein was prepared for docking as follows: water molecules were ignored; hydrogen atoms were added; and the co-crystallized ligand was used to determine the binding pocket. The compounds were drawn on ChemDraw and transferred to Discovery Studio (Table S1).

Prediction of Pharmacokinetics Properties and Drug Likeliness
SwissADME server-a free web tool (http://www.swissadme.ch/index.php (accessed on 10 November 2021) developed by Swiss Institute of Bioinformatics-was utilized to compute physicochemical descriptors as well as to predict ADME parameters, pharmacokinetic properties, druglike nature, and the medicinal chemistry friendliness of compounds 6a-m to support drug discovery [86].

Conclusions
A novel set of quinoline-based uracil hybrids has been tailored and synthesized. The ability of the later hybrids 10a-l toward inhibition of hCA I, II (cytosolic) and hCA IX, XII (transmembrane, tumor-associated isoforms) using colorimetric assay was evaluated. The results revealed that our novel hybrids 10a-l had selective inhibition of hCA IX and XII comparable with hCA I and II in the micromolar range. Hybrids 10d and 10l have been carefully selected, as optimal compounds for higher hCA IX inhibitory activity and selectivity, for further investigation of their antiproliferative activity against the breast cancerous cell line MCF-7 and the lung cancer cell line A549 using the MTT protocol, which was comparable to the reference standard staurosporine. Compound 10d had a superior inhibition of MCF-7 cells than A549 cells with IC 50 = 2.87 ± 0.05 and 11.83 ± 0.22, respectively. Similarly, the hybrid 10l displayed stronger inhibition of MCF-7 than of A549 with IC 50 = 4.08 ± 0.08 and 26.10 ± 0.56, respectively. Further, both hybrids induced apoptosis in MCF-7 and A549 cells, together with worthy and desirable changes in Bax/Bcl expression ratio. The modeling study displayed high docking scores and good binding interactions of the most active compounds, 10d and 10l, within the hCA-IX active pocket, with adoption of orientation similar to that of co-crystalized ligand. This highlights our proposition of the impact of the electron-rich environment of the sulfur atom on the uracil backbone, as well as the insertion of small lipophilic and non-sterically hindered groups on both quinoline and uracil pharmacophores as crucial features for accessing highly selective hCA IX and XII inhibitors. Overall, our novel hybrids have opened the door to a new authentic approach for the engaging the quinoline scaffold with the uracil pharmacophore, which is a tactic that has rarely been discussed to date. Indeed, compounds 10d and 10l are likely to be potential lead candidates for further investigation and optimization, i.e., the rational development of novel, potent tumor-associated hCAs IX and XII selective inhibitors as agents for cancer treatment.