Biaryl Sulfonamides Based on the 2-Azabicycloalkane Skeleton—Synthesis and Antiproliferative Activity

In a search for new, selective antitumor agents, we prepared a series of sulfonamides built on bicyclic scaffolds of 2-azabicyclo(2.2.1)heptane and 2-azabicyclo(3.2.1)octane. To this end, aza-Diels–Alder cycloadducts were converted into amines bearing 2-azanorbornane or a bridged azepane skeleton; their treatment with sulfonyl chlorides containing biaryl moieties led to the title compounds. The study of antiproliferative activity of the new agents showed that some of them inhibited the growth of chosen cell lines with the IC50 values comparable with cisplatin, and some derivatives were found considerably less toxic for nonmalignant cells.


Introduction
The development of pharmaceuticals used for cancer treatment is connected with an introduction of various classes of antineoplastic drugs: metal complexes, alkylating agents, antimetabolites, natural products, etc. The success of certain chemotherapeutics like cisplatin is undoubtful, but side effects and drug resistance problems prompt chemists to search for new alternatives with comparable activity against malignant cells, but increased selectivity [1,2]. Sulfonamides, generally associated with their traditional use as antibacterial drugs, were found to exhibit a multidirectional biological activity [3,4], including the ability to inhibit the growth of selected tumors [5][6][7][8][9]. Besides the main functional group present in the molecule, also certain structural motives were identified as beneficial for the desired therapeutic action. Biphenyl, or, more general, biaryl moiety is found in many natural and synthetic compounds of biomedical interest. As an example, Luo and co-workers identified two new ATP-competitive inhibitors of kinesin spindle protein which plays an essential role in the early stages of mitosis [10]. These compounds containing a biphenyl fragment appeared efficient against colorectal cell line. Based on this discovery, urea and thiourea derivatives bearing this substituent were tested in vitro by Holland, Fischer, and co-workers using prostate, ovarian, and breast cancer cells, with three compounds showing desired activity and selectivity [11]. In another study by Zhang and co-workers, biphenyl-based ureas displayed potent ability to inhibit the proliferation of human lung cancer cells A549 and human hepatoma cells [12]. One of biphenyl methylene indolones tested by Donthiboina et al. was found a particularly efficient growth inhibitor of HeLa and prostate cell line DU-145 [13].
In our research, we focus on a preparation and applications of multifunctionalized compounds based on a chiral, bicyclic skeleton. 2-azabicyclo(2.2.1)heptane (2-azanorbornane), synthetically available through a stereoselective aza-Diels-Alder reaction, is regarded as a versatile platform for the synthesis of a variety of derivatives for the use in asymmetric synthesis, but also biomedical studies [14]. Importantly, due to the multiplication of chirality in the cycloaddition step, a single enantiomer bearing four stereogenic centers with a defined configuration can be isolated [14][15][16][17][18][19]. The skeleton of the base compound can be appropriately modified either by introduction of various groups, typically into position 3 or 2, formation of dimeric species, and extending of the bicyclic system (in a stereoselective manner) to 2-azabicyclo(3.2.1)octane [20]. Certain functionalities, like hydroxyl or amine groups, offer a convenient access to a set of other derivatives; on the other hand, the presence of the amine moiety opens the possibility of interactions (e.g., by the formation of hydrogen bonds) in a biological system.
In our previous study on the biomedical applications of 2-azabicycloalkane derivatives a series of sulfonamides based on the bicyclic chiral scaffold were prepared [21]. The cytotoxicity of the synthesized, stable products was tested using human cancer cell lines: glioblastoma (GBM) medulloblastoma (MB), and hepatocellular carcinoma (HCC), and several sulfonamides were found to exhibit significant cytotoxicity. However, as we concluded, discrimination of toxicity between malignant and nonmalignant cells was too narrow (less than >30-fold difference which is suggested on security grounds [22]). Thus, we decided to introduce appropriate modifications to the structures of the sulfonamides, including substituents of SO 2 fragment, but also at N-2 of the bicyclic system, and the linker between the skeleton and a functional group. The results of our exploration are presented in this contribution.

General Considerations
All the solvents and reagents were received from chemical companies and we used them without additional purification. Schmelzpunkt Bestimmer Apotec melting point apparatus (WEPA Apothekenbedarf GmbH & Co. KG., Hillscheid, Germany) was used for the determination of melting points in a standard open capillary. 1 H and 13 C NMR spectra were recorded on Jeol 400yh (Jeol Ltd., Tokyo, Japan), Bruker Avance III500, and Bruker Avance II 600 spetrometers (Bruker, Billerica, MA, USA). The residual 1 H or 13 C signals of the solvent (chloroform-d) were used as references. Chemical shifts are given in ppm, and coupling constants are expressed in Hz. The spectra are shown in Figures S1-S18. High-resolution mass spectra were recorded using electrospray ionization mode on a Waters LCT Premier XE TOF spectrometer (Waters Corporation, Milford, MA, USA). Infrared spectra were measured in a 4000-400 cm −1 range on a Perkin Elmer 2000 FTIR instrument (PerkinElmer, Waltham, MA, USA). Optical rotations were determined with an automatic polarimeter Model AA-5 (Optical Activity, Ltd., Ramsey, UK); [α] D values are expressed in 10 −1 deg cm 2 g −1 . Silica gel 60 (60-200 µm, 70-230 mesh) was used for column chromatography, and precoated plates precoated with the same adsorbent were apllied for thin-layer chromatography.
Four-circle single crystal diffractometer (Oxford Diffraction Ltd., Wrocław, Poland) with a CCD Atlas detector using graphite-monochromatized MoK α radiation (λ = 0.71073 Å) was applied for the collection of X-ray diffraction data. The raw data were processed with the CrysAlis Data Reduction Program (version 1.171.39.46). Corrections for polarization and Lorentz effects were introduced to reflection intensities. The crystal structure was solved by direct methods with SHELXS-2018/3 and refined using full-matrix least-squares method using SHELXL-2018/3 program [23]. Anisotropic displacement parameters were applied for the refinement of non-hydrogen atoms. H-atoms, though visible on the Fourier difference maps, were placed by geometry and allowed refined 'riding on' the parent atom with U iso = 1.2 U eq (C) for CH and CH 2 groups and U iso = 1.5 U eq (C) for CH 3 groups. Coordinates of hydrogen atom of the N-H group was refined for 9i, but U iso = 1.2 U eq (N). In the case of compound 10i, hydrogen atom position of the N-H groups were fixed at values found for maximum at the difference map and U iso = 1.5 U eq (N). The reflection intensities were treated by the PLATON program (version 281019) with 'squeeze' procedure, because the position of a solvent molecule was not determined. The details of crystal data and structure refinement are presented in Table S1. Structures were visualized using Diamond 3.2k [24].  [20,25,26]. Preparation of enantiomerically pure 2-azanorbornane amine derivatives with two bicyclic subunits was described as well [25]. Amine 8 was prepared from (1S,4S,5R)-4-chloro-2-((S)-1-phenylethyl)-2-azabicyclo(3.2.1)octane [20] via the reaction with potassium cyanide followed by reduction with LiAlH 4 (yield 64%). Amine 5 was obtained using an adapted procedure from the patent [27].

General Procedure for Sulfonamide Synthesis
Primary amine (1.0 mmol) and KOH (powdered, 0.10 g, 1.8 mmol, 1.8 equiv.) were dissolved in dry CH 2 Cl 2 (15.0 mL). A chosen sulfonyl chloride (1.0 mmol, 1.0 equiv.) was then introduced. The mixture was stirred for 24 h at room temperature. After addition of brine the mixture was extracted with dichloromethane. The organic phases were dried (Na 2 SO 4 ), and evaporated to dryness after filtration through Celite ® . Thus obtained crude sulfonamides were purified using column chromatography on silica, and eluted with ethyl acetate/n-hexane (1: The details of experimental procedures and the relevant physicochemical data of all newly prepared compounds together with copies of 1 H and 13 C NMR spectra are gathered in Supplementary Materials.

Cell Culture
The following human cancer cell lines, representing the most common types of cancer, were chosen for evaluation of the antiproliferative activity of the prepared sulfonamides: lung cancer (A549), breast cancer (MCF-7), colon adenocarcinoma (LoVo), and biphenotypic B cell myelomonocytic leukemia (MV4-11). As a reference, the normal mouse fibroblasts cell line (BALB/3T3) was applied. A549, LoVo and BALB/3T3 cell lines were purchased from the ATCC (American Type Culture Collection, Rockville, MD, USA), MCF-7 cell line-from EACC (The European Collection of Cell Cultures), and MV4-11 cell line was obtained from DSMZ (Leibniz Institute-German Collection of Microorganisms and Cell Culture, Braunschweig, Germany). All these lines were maintained at the Hirszfeld Institute of Immunology and Experimental Therapy (IIET) in Wrocław, Poland.

In Vitro Anti-Proliferative Assays
Twenty-four hours before addition of the tested compounds, each cell line was seeded in 96-well plastic plates (Sarstedt, Numbrecht, Germany) in an appropriate medium at 10 4 cells/well density, except MCF7 cell line: 0.75 × 10 4 /well, and A549 cell line: 0.25 × 10 4 /well. The studied cell lines were subjected to each of the tested sulfonamides at 4 different concentrations in the range of 100-0.1 µg/mL for 72 h. Cisplatin (Teva Pharmaceuticals, Poland) was used as a reference compound, and DMSO (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) served as a solvent control at concentrations equivalent to these applied in the solutions of the tested sulfonamides. MTT assay was performed for leukemic cells, while sulforhodamine B assay (SRB) for the adherent cells.

SRB Assay
After 72 h of incubation, cells were fixed in situ by gently adding of 50 µL per well of ice-cold 50% TCA (trichloroacetic acid, POCh, Gliwice, Poland) and were incubated at 4 • C for one hour. Each well was then washed five times with water, followed by addition of 50 µL of 0.4% solution of SRB (sulforhodamine B, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 1% acetic acid (POCh, Gliwice, Poland), and plates were again incubated at RT for 0.5 h. Plates were washed five times with 1% acetic acid to remove the unbound dye. The stained cells were treated with 10 mM TRIS (Tris base, Sigma-Aldrich, Chemie GmbH, Steinheim, Germany). Absorbance at 540 nm in each well was read with Elisa plate reader (BioTek Synergy H4, Swindon, UK) using the Gen5 software [28].
The results are shown as average IC 50 values (concentration of the compound causing inhibition of cell proliferation by 50%) ± standard deviation. IC 50 values were determined using the Prolab-3 system based on Cheburator 0.4, a software developed by Nevozhay [30]. At each concentration, sulfonamides were tested in triplicates in a single experiment. Three independent repetitions were applied for each experiment.
The protocol started from a stereoselective reaction between cyclopenatdiene and an enantiopure imine (prepared in situ from (S)-1-phenylethylamine and ethyl glyoxylate); the major product, exo isomer 1, was isolated and used in further transformations (Scheme 1) [14][15][16][17][18][19]. Its reduction, depending on conditions applied, led either to alcohol 2 with a preserved N-substituent, or to product 3 in which the group was removed and replaced with Boc protection (Scheme 1). In the case of derivative 2 its reaction with azide under Mitsunobu conditions resulted in ring expansion, yielding-after reduction-a bridged azepane bearing amine function 4 [20]. The reaction was shown to proceed through aziridinium intermediate formed by the attack of N-2 nucleophile on a carbon atom of the substituent in position 3. In contrast, compound 3 underwent a smooth nucleophilic substitution without alteration of the bicyclic system (Scheme 2). This effect can be attributed to the electron withdrawing properties of tert-butoxycarbonyl group which decreases the nucleophilic character of N-2 center. Thus obtained azide was readily converted to the corresponing amine 5. Its isomer with a 2-azabicyclo(2.2.1.)heptane scaffold 6 was prepared as described in our prevoious works: Swern oxidation followed by transformation of the formed aldehyde to oxime and its reaction with LiAlH 4 [27]. Finally, a homologue of amine 4 with an additional methylene group in the substituent was obtained from alcohol 2 in three steps: reacton with sulfonyl chloride in the presence of pyridine resulted in ring expansion and introduction of Cl substituent which was then replaced with cyanide to afford nitrile 7. Its reduction provided amine 8 which combined structural features of two isomeric compounds 4 and 6: an enlarged ring and amino group attached to it through a flexible spacer. Having in hand four amines (4, 5, 6, and 8), we reacted them with chosen sulfonyl chlorides, affording coresponding sulfonamides in 70-80% yield in most cases (Scheme 3). The choice of reagents used was based on the results of our previous investigations as well as literature precedents. We expected that introducing biaryl substituents, and, in particular, fluorinated biaryls, should result in an enhanced antiproliferative activity.
All new compounds were fully characterized using spectroscopic methods (IR, 1 H and 13 C NMR, HRMS). In addition, for two fluorinated derivatves, 9i and 10i, we obtained crystals suitable for Xray diffraction measurements; their structures further confirmed the formation of the expected products bearing four (9i) or three stereogenic centers (10i) of defined configuration: (1S,4S,5R,1'S) for 9i and (1S,3R,4R) in the case of 10i. Having in hand four amines (4, 5, 6, and 8), we reacted them with chosen sulfonyl chlorides, affording coresponding sulfonamides in 70-80% yield in most cases (Scheme 3). The choice of reagents used was based on the results of our previous investigations as well as literature precedents. We expected that introducing biaryl substituents, and, in particular, fluorinated biaryls, should result in an enhanced antiproliferative activity.
All new compounds were fully characterized using spectroscopic methods (IR, 1 H and 13 C NMR, HRMS). In addition, for two fluorinated derivatves, 9i and 10i, we obtained crystals suitable for Xray diffraction measurements; their structures further confirmed the formation of the expected products bearing four (9i) or three stereogenic centers (10i) of defined configuration: (1S,4S,5R,1'S) for 9i and (1S,3R,4R) in the case of 10i. Having in hand four amines (4, 5, 6, and 8), we reacted them with chosen sulfonyl chlorides, affording coresponding sulfonamides in 70-80% yield in most cases (Scheme 3). The choice of reagents used was based on the results of our previous investigations as well as literature precedents. We expected that introducing biaryl substituents, and, in particular, fluorinated biaryls, should result in an enhanced antiproliferative activity.
All new compounds were fully characterized using spectroscopic methods (IR, 1 H and 13 C NMR, HRMS). In addition, for two fluorinated derivatves, 9i and 10i, we obtained crystals suitable for X-ray diffraction measurements; their structures further confirmed the formation of the expected products bearing four (9i) or three stereogenic centers (10i) of defined configuration: (1S,4S,5R,1'S) for 9i and (1S,3R,4R) in the case of 10i.

Scheme 3. Preparation of sulfonamides 9-12.
The compounds 9i and 10i crystallize in enantiomorphic P21 and I2 space groups, respectively. The crystal structures are non-centrosymmetric, because they consist of only one enantiomer. Selected bond lengths and angles are presented in Table S2. The values are normal and show a well established atomic framework. Geometry parameters for hydrogen bonds indicate that weak intermolecular interactions exist between adjacent molecules (Table S3). The most relevant base on the N-H group, which forms N-H···O hydrogen bond with oxygen atom of the SO2 group in 9i. In the case of 10i, the nonsubstituted nitrogen atom of the 2-azanorbornane skeleton is an acceptor in N-H···N hydrogen bond. These two types of interactions appear to be of high importance in a view of molecular self-assembly, as they connect two symmetry independent molecules in the crystal structure of 9i and 10i and arrange the molecule in dimers (Figures 1 and 2). In the crystal structure of 10i, the dimers are additionally connected to each other by the N-H···O hydrogen bonds, which stabilize the crystal structure along b crystallographic direction. In dimers of 9i and 10i, two hydrogen bonds form ring patterns R 2 2(8) and R 2 2(10), which occur around the non-crystallographic inversion center [31,32].

Scheme 3. Preparation of sulfonamides 9-12.
The compounds 9i and 10i crystallize in enantiomorphic P2 1 and I2 space groups, respectively. The crystal structures are non-centrosymmetric, because they consist of only one enantiomer. Selected bond lengths and angles are presented in Table S2. The values are normal and show a well established atomic framework. Geometry parameters for hydrogen bonds indicate that weak intermolecular interactions exist between adjacent molecules (Table S3). The most relevant base on the N-H group, which forms N-H···O hydrogen bond with oxygen atom of the SO 2 group in 9i. In the case of 10i, the nonsubstituted nitrogen atom of the 2-azanorbornane skeleton is an acceptor in N-H···N hydrogen bond. These two types of interactions appear to be of high importance in a view of molecular self-assembly, as they connect two symmetry independent molecules in the crystal structure of 9i and 10i and arrange the molecule in dimers (Figures 1 and 2). In the crystal structure of 10i, the dimers are additionally connected to each other by the N-H···O hydrogen bonds, which stabilize the crystal structure along b crystallographic direction. In dimers of 9i and 10i, two hydrogen bonds form ring patterns R 2 2 (8) and R 2 2 (10), which occur around the non-crystallographic inversion center [31,32].

Antiproliferative Activity of Tested Compounds
Antiproliferative activity of enantiomerically pure sulfonamides 9-12 was investigated using human acute myeloid leukemia cell line (MV4-11) and three solid tumor cell lines: lung (A549), colorectal (LoVo), and breast (MCF-7). Normal murine fibroblasts cell line (BALB/3T3) was used for the evaluation of selectivity of the inhibitors. The results are collected in Table 1.
Analysis of the results obtained for a full series of sulfonamides based on one scaffold of 2-azanorbornane (11a-j) provided a valuable insight into an impact of substitution pattern on the observed antiproliferative action. Among the tested sulfonamides, derivatives 11a-c bearing methyl, phenyl, and tolyl substituents were found practically inactive in concentrations up to 100 µM or exhibited only moderate activity against leukemia cell line (11b,11c). Introduction of fluorine substituent did not lead to a considerable improvement (compound 11d). However, trifluoromethyl derivative 11e was able to inhibit proliferation of all cell lines, with the most promising result for MV4-11 (16.15 ± 4.95 µM). Still, the results for this sulfonamide were significantly surpassed by biaryl-substituted compounds, in agreement with our expectations. All five derivatives 11f-j showed a desired activity, in some cases with IC 50 value comparable with cisplatin. In particular, the results obtained for compounds 11f (3.09 ± 1.56 µM) and 11g (4.26 ± 3.97 µM) are amongst the best in the whole series. On the other hand, the same sulfonamides were much worse in comparison with 11h-11j in the growth inhibition of breast cancer MCF-7. Fluorinated derivative, 11i, as expected, exhibited a reasonable antiproliferative activity and all IC 50 values were smaller than 15 µM; the best value 2.98 ± 1.38 µM for A549 line was the lowest among all sulfonamides tested. One should not forget, however, that in many cases a proven performance against cancer cells is accompanied with high toxicity for normal cells (vide infra).  Comparison of compounds bearing the same fluorinated biphenyl substituent, namely 9i, 10i, 11i, and 12i revealed the influence of the chiral bicyclic scaffold on the utility of these azabicyclic compounds as the possible drug candidates. As it can be seen in Table 1, ring size and flexibility of the connection does not change much the effect of application of given sulfonamide for all tested lines. All fluorinated derivatives exhibited a significant activity comparable with cisplatin, and even in some cases the IC 50 values were lower (derivative 11i, A548 and LoVo lines). The presence of N-2 (1-phenylethyl) substituent was generally beneficial since the activity of 10i, capable of formation of additional hydrogen bonds (as exemplified by its X-ray structure) was decreased in comparison to 11i, with an exception of MV4-11 leukemia cells. The results for isomers (9i vs. 11i) and homologs (12i vs. 11i) suggested that a bigger bicyclic system with amino group attached directly to it is slightly more active (meaningful differences were found for A549 and LoVo lines).

Antiproliferative Activity of Tested Compounds
Antiproliferative activity of enantiomerically pure sulfonamides 9-12 was investigated using human acute myeloid leukemia cell line (MV4-11) and three solid tumor cell lines: lung (A549), colorectal (LoVo), and breast (MCF-7). Normal murine fibroblasts cell line (BALB/3T3) was used for the evaluation of selectivity of the inhibitors. The results are collected in Table 1.  Finally, we can analyze the effect of substitution of the biaryl system (compounds 9f, 9h, and 9i and 11f-11j). While for the 2-azabicyclo(3.2.1.)octane system the presence of methoxy group resulted in a slight decrease of IC 50 values (9f vs. 9h), for 2-azanorbornane derivatives the impact of -OCH 3 fragment varied with the cell line (10f vs. 10h). Similarly, methyl substituent decreases the activity for A549 cells, but increases for LoVo and MCF-7 (10f vs. 10g). Fluorine in biphenyl system of 11 was found beneficial for the treatment of solid tumors (but not for leukemia cells), and placement the halogen in para position seemed better in that case (11i vs. 11j).
Additionally, the results for dimeric species 13f and 13i, bearing biphenyl and fluorobiphenyl substituent, respectively ( Figure 3) were, to our surprise, practically inactive against the tested cell lines, with 13i leading to even worse results.
Materials 2020, 13, x FOR PEER REVIEW 9 of 12 Analysis of the results obtained for a full series of sulfonamides based on one scaffold of 2azanorbornane (11a-j) provided a valuable insight into an impact of substitution pattern on the observed antiproliferative action. Among the tested sulfonamides, derivatives 11a-c bearing methyl, phenyl, and tolyl substituents were found practically inactive in concentrations up to 100 µM or exhibited only moderate activity against leukemia cell line (11b,11c). Introduction of fluorine substituent did not lead to a considerable improvement (compound 11d). However, trifluoromethyl derivative 11e was able to inhibit proliferation of all cell lines, with the most promising result for MV4-11 (16.15 ± 4.95 µM). Still, the results for this sulfonamide were significantly surpassed by biarylsubstituted compounds, in agreement with our expectations. All five derivatives 11f-j showed a desired activity, in some cases with IC50 value comparable with cisplatin. In particular, the results obtained for compounds 11f (3.09 ± 1.56 µM) and 11g (4.26 ± 3.97 µM) are amongst the best in the whole series. On the other hand, the same sulfonamides were much worse in comparison with 11h-11j in the growth inhibition of breast cancer MCF-7. Fluorinated derivative, 11i, as expected, exhibited a reasonable antiproliferative activity and all IC50 values were smaller than 15 µM; the best value 2.98 ± 1.38 µM for A549 line was the lowest among all sulfonamides tested. One should not forget, however, that in many cases a proven performance against cancer cells is accompanied with high toxicity for normal cells (vide infra).
Comparison of compounds bearing the same fluorinated biphenyl substituent, namely 9i, 10i, 11i, and 12i revealed the influence of the chiral bicyclic scaffold on the utility of these azabicyclic compounds as the possible drug candidates. As it can be seen in Table 1, ring size and flexibility of the connection does not change much the effect of application of given sulfonamide for all tested lines. All fluorinated derivatives exhibited a significant activity comparable with cisplatin, and even in some cases the IC50 values were lower (derivative 11i, A548 and LoVo lines). The presence of N-2 (1-phenylethyl) substituent was generally beneficial since the activity of 10i, capable of formation of additional hydrogen bonds (as exemplified by its X-ray structure) was decreased in comparison to 11i, with an exception of MV4-11 leukemia cells. The results for isomers (9i vs 11i) and homologs (12i vs. 11i) suggested that a bigger bicyclic system with amino group attached directly to it is slightly more active (meaningful differences were found for A549 and LoVo lines).
Finally, we can analyze the effect of substitution of the biaryl system (compounds 9f, 9h, and 9i and 11f-11j). While for the 2-azabicyclo(3.2.1.)octane system the presence of methoxy group resulted in a slight decrease of IC50 values (9f vs. 9h), for 2-azanorbornane derivatives the impact of -OCH3 fragment varied with the cell line (10f vs. 10h). Similarly, methyl substituent decreases the activity for A549 cells, but increases for LoVo and MCF-7 (10f vs. 10g). Fluorine in biphenyl system of 11 was found beneficial for the treatment of solid tumors (but not for leukemia cells), and placement the halogen in para position seemed better in that case (11i vs. 11j).
Additionally, the results for dimeric species 13f and 13i, bearing biphenyl and fluorobiphenyl substituent, respectively ( Figure 3) were, to our surprise, practically inactive against the tested cell lines, with 13i leading to even worse results. Several conclusions on key structural features can be drawn from such a preliminary SAR (structure-activity relationship) analysis. Biaryl substituent on sulfonamide and N-(1-phenylethyl) group can be regarded as prerequisites for the desired activity. To discuss further the effect of introduction of fluorine, we should take into account the selectivity of the tested compounds.   Figure S1.