European Journal of Medicinal Chemistry 101 (2015) 358e366

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis of bicoumarin thiophosphate derivatives as steroid sulfatase inhibitors Sebastian Demkowicz a, *, Witold Kozak a, Mateusz Da
sko a, Maciej Masłyk b, Bartłomiej Gielniewski b, Janusz Rachon a a

Department of Organic Chemistry, Chemical Faculty, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland
w 1i, Department of Molecular Biology, Faculty of Biotechnology and Environment Sciences, The John Paul II Catholic University of Lublin, Konstantyno 20-708 Lublin, Poland b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 November 2014 Received in revised form 26 June 2015 Accepted 26 June 2015 Available online 2 July 2015

Based on the frameworks of 7-hydroxy-2,3-dihydro-1H-cyclopenta[c]chromen-4-one, 3-hydroxy7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one and 3-hydroxy-8,9,10,11-tetrahydro-7H-cyclohepta[c] chromen-6-one, a series of bicoumarin thiophosphate analogs have been synthesized and biologically evaluated. Additionally, their binding modes have been modeled using docking techniques. The inhibitory properties of the synthesized compounds were tested against the STS isolated from human placenta. Most of the new STS inhibitors possessed good activities against STS. In particular, we found that the bis(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl) hydrogenthiophosphate (10b) produced the largest inhibitory effect, with an IC50 value of 860 nM (an IC50 value of 1 mM for the 665-COUMATE used as a reference). The structure-activity relationships of the synthesized bicoumarin thiophosphate derivatives toward the STS enzyme have been discussed previously. © 2015 Published by Elsevier Masson SAS.

Keywords: Steroid sulfatase STS inhibitors Breast cancer Bicoumarin thiophosphates Molecular docking

1. Introduction Biologically active hormones, including androgens and estrogens, play an important role in the development of many diseases, such as hormone-dependent breast cancer (HDBC). One approach for the treatment of HDBC is based on the inhibitors of the steroid sulfatase (STS) [1]. The STS is responsible for the hydrolysis of steroid sulfates into their active forms and thus plays a crucial role in the formation of biologically active steroids. The STS hydrolyses, among others, estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) into estrone (E1) and dehydroepiandrosterone (DHEA), respectively, which can be converted into steroids that exhibit estrogenic properties (estradiol or androstenediol) [2]. The wide distribution of the STS enzyme throughout the body is an indication of its involvement in numerous physiological and pathological conditions [3]. Although the crystal structure of the STS has been determined [4], relatively little is known about the regulation of its expression and activity. Because of the close relationship between the steroid sulfatase

* Corresponding author. E-mail address: [email protected] (S. Demkowicz). http://dx.doi.org/10.1016/j.ejmech.2015.06.051 0223-5234/© 2015 Published by Elsevier Masson SAS.

and the arylsulfatases A and B, the topology of the active site of all three enzymes is similar. A characteristic feature of all sulfatases is the exposure of a posttranslational modification that involves the conversion of the cysteine residue in the active site of the enzyme into a formylglycine (FGly) residue [5]. In the resting state, the active site of the human STS consists of a sulfated FGly residue in its gem-diol form, which is coordinated to a Ca2þ cation. The catalytic region of the STS is also formed by nine other catalytically important amino acid residues: Asp35, Asp36, Arg79, Lys134, His136, His290, Asp342, Gln343, and Lys368. Furthermore, when the natural substrate, e.g., E1S, is located in the active site of the STS, the Leu74, Arg98, Thr99, Val101, Leu103, Leu167, Val177, Phe178, Thr180, Gly181, Thr484, and Phe488 amino acid residues surround and interact with the steroidal core of the substrate via an hydrophobic interaction [3]. Taking into account that the unsulfated FGly residue in its gemdiol form plays a crucial role in the hydrolysis of the sulfate substrates by the arylsulfatase A and B [6], the putative mechanism of action of the STS is shown the in Fig. 1. First, the formation of the unsulfated FGly residue (gem-diol) from the structure containing the sulfate moiety (FGlyS) occurs. This step can be performed via the desulfation of the FGlyS, which is catalyzed by the nonesterified hydroxyl group, followed by the nucleophilic attack of the water

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Fig. 1. Putative mechanism of action of the STS (E e enzyme).

molecules on the carbon atom of the FGly intermediate (pathway A). The pathway B assumes a direct nucleophilic substitution reaction on the sulfur atom of the FGlyS. Finally, an SN2 attack of the hydroxyl group of the FGly (gem-diol) on the sulfur atom of the substrate (e.g., E1S), hydrolyzes the conjugate alcohol and creates a sulfated FGly (FGlyS) [3]. The design and synthesis of new and more effective agents that inhibit the activity of the STS is a major challenge for modern medicinal chemistry. Most of the STS inhibitors discovered to date act in an irreversible way. One of the first irreversible inhibitors was the EMATE (1), which exhibits a very high activity in MCF-7 cells

and has an IC50 value of 65 pM [7] (Fig. 2). Despite the exceptional potency of the EMATE, it is not used to treat hormone-dependent breast cancer because of its estrogenic properties [8]. It has been known for a long time that the nonsteroidal agents and their metabolites, which were designed for potential therapeutic use, induce fewer undesirable endocrine effects in vivo than do their steroidal analogues. The attempts to synthesize non-estrogenic compounds have promoted the generation of the tricyclic coumarin sulfamates. Among them, the compound 667 COUMATE (2) (currently in clinical trials) was shown to be a potent STS inhibitor, with an IC50 value of 8 nM in placental

Fig. 2. Chemical structure of the STS inhibitors.

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microsomes. The compound 667 COUMATE (2) did not display any estrogenic activity in in vitro and in vivo assays [9]. Other promising inhibitors that do not exhibit undesirable estrogenic effects are the STX213 (3) and STX1938 (4). The STX213 (3), a natural steroid that contains an N-substituted piperidine-2,6-dione ring, was shown to be a more potent STS inhibitor in rat liver than the 667 COUMATE (2), and exhibited a greater efficacy than the 667 COUMATE (2) evaluated on MCF-7STS xenografts in mice [10]. The STX1938 (4) displayed a similar STS inhibitory activity compared with the STX213 (3) [11]. In parallel to the development of the 667 COUMATE 2, scientists from Kyowa Hakko Kogyo Co. Ltd reported a series of 17beta-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives as potent and selective STS inhibitors [12]. Among them, the compound KW-2581 (5) inhibited the STS activity with an IC50 of 4.0 nM when evaluated on the crude enzymes isolated from recombinant Chinese hamster ovary cells, which express the human arylsulfatases (ARSs) and are very promising therapeutic targets for clinical trials [13]. The goal of our study was to design new STS inhibitors based on bicoumarin scaffolds. The coumarins are an important class of naturally occurring compounds and have been reported to exhibit a variety of biological activities. In addition to the aforementioned role of the coumarins as inhibitors of the STS, they have been recognized to possess antioxidant [14], antiasthmatic [15], antiproliferative [16], anticancer, or antimalarial [17] activities. Our particular scientific interests are focused on the use of different thiophosphate moieties in the design of novel STS inhibitors. We found that these compounds are stable (even in aqueous solutions) and readily available and that, as is widely recognized, they may undergo a nucleophilic substitution reaction on the phosphorus atom or create many interactions (e.g., hydrogen bonds) with a variety of amino acid residues found within the active site of the STS. Additionally, the bicoumarin scaffolds exhibit different docking modes (not previously described) and seem to be very promising as a new type of STS inhibitors.

favorable in the case of the bicoumarin chlorothiophosphates 8ae8c and the bicoumarin hydrogenthiophosphates 10ae10c, which led to values within the lowest free binding energy range (between 5.2 and 8.1 kcal mol1). Fig. 3 shows a putative enzyme-ligand complex before the presumed inactivation of the STS and the superimposed best conformations of the three bicoumarin hydrogenthiophosphates 10ae10c. As shown in Fig. 3, the designed STS inhibitors exhibited a slightly different docking compared with the reported mode of the sulfamate-based STS inhibitor (665-COUMATE). In this case, the skeleton of one of the nonpolar tricyclic coumarins was oriented in the center of the active site and underwent a non-polar interaction with the side chains of the hydrophobic pocket formed by the Arg98, Val101, Leu103, Leu167, Val177, Phe178, His485, Val486, and Phe488 residues. The other parts of the bicoumarin scaffolds are located outside the entrance of the active site of the STS. Generally, the designed STS inhibitors can adopt conformations that are able to fill the whole cavity and prevent the substrate to access the catalytic residues. It is worth noting that the results of the docking studies predicted that the compounds with six-membered rings would have the best binding positions toward the STS in terms of docking energy (for example, the value of the Autodock Vina score for 10b was 8.1 kcal mol1 and was in agreement with the experimental data of the STS enzyme assays). When studying the inhibitorenzyme interactions at the atomic level, it can be observed that the thiophosphate moieties of the 10ae10c compounds are directed toward the Thr484 residue and that their OH groups are able to establish hydrogen bonds (see Fig. 4). Generally, the identified hydrogen bond between the thiophosphate moieties and the Thr484 residue could favor the binding and may have a significant impact on the enzyme-ligand complex stability. Furthermore, in the case of the compounds 8ae8c, the arrangement of the thiophosphate moiety and its distance to the hydroxyl group of Thr484 suggest that a thiophosphate group transfer may be crucial during the inactivation process.

2. Results and discussion

2.2. Chemistry

2.1. Molecular modeling

The bicoumarins are a large group of naturally occurring oxygen heterocycles that are distributed extensively among various types of flora [18]. Most of them are bridged through CeOeC and CeC linkage. It is well known that the bicoumarins are naturally formed as a result of the free radical oxidative coupling of monomeric aromatic precursors [19]. Because of their high biological activity and use in the design of new drugs, their synthesis has captured the interest of many investigators. Many synthetic methods for the preparation of the bicoumarin exist, which include the reaction of 4-hydroxycoumarin with substituted Meldrum acids [20], the Knoevenagel condensation of (coumarin-4-yl)acetic acids and their esters with substituted salicylic aldehydes [21], and the oxidative coupling of 7-methoxy-4-methyl coumarin with Mn(OAc)3/HCIO4 [22]. Other oxidative coupling conditions have been used in the multistep synthesis of naturally occurring bicoumarins, such as the Büchi synthesis of racemic kotanin (oxidation of the organolithium derivative with cupric chloride) [23], the Lin asymmetric synthesis of the kotanin (oxidative coupling using CuCN/TMEDA) [24], the Hüttel total synthesis of kotanin, isokotanin A, and desertorin C using oxidative phenol coupling (FeCl3/SiO2) [25], and the Bringmann atropo-enantioselective synthesis of (D)- isokotanin A (oxidative coupling with Cu/DMF) [26]. As a result of the previous theoretical analyses, we decided to synthesize bicoumarin thiophosphate derivatives based on tricyclic coumarin analogs 6a, 6b and 6c. The synthesis of tricyclic coumarin scaffolds has been performed via the Pechmann condensation from corresponding b-ketoesters and resorcin in the presence of

The X-ray structure of the human steroid sulfatase was retrieved from the Protein Databank (Protein Data Bank accession code 1P49) and prepared for docking using the following procedure: 1) the waters of crystallization were removed from the structure; 2) the catalytic amino acid FGly75 (N-formylglycine) was converted to the gem-diol form using the Protein Preparation Wizard module €dinger, LLC, New York, NY); 3) delivered with Maestro (Schro hydrogen atoms were added to the structure and the entire molecule was subjected to a minimization using the OPLS-AA force field. Before the docking calculations, the inhibitors were constructed using the Portable HyperChem 8.0.7 software (Hypercube Incorporation). Each inhibitor was optimized using the MM þ force
re conjugate gradient algorithm. The iterfield and the Polak-Ribie ative procedure was continued until the energy gradient became lower than 0.1 kcal/mol/Å. The docking of the optimized inhibitors into the prepared structure of the human steroid sulfatase was performed using the Autodock Vina 1.1.2 software (The Molecular Graphics Laboratory, Scripps Research Institute). For all the docking studies, a grid box size of 30 Å  30 Å  30 Å centered on the Cb atom of the amino acid 75 was used. The graphic visualizations of the 3D model were generated using the VMD 1.9 software (University of Illinois at UrbanaeChampaign). The docking experiments were performed 20 times for each compound. The predicted free binding energies were more

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Fig. 3. Docked binding modes for the compounds 10a (blue), 10b (yellow), 10c (green), and 665 COUMATE (black). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Docked binding modes for compounds 8b (purple), with distances between Thr484 and the phosphorus atom, and 10b (yellow), with the predicted hydrogen bond. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

concentrated H2SO4 as an acidic catalyst [27]. The Pechmann condensation is one of the most common procedures for the preparation of coumarin and its derivatives. In the case of substituted phenols, the electron-donating groups are required for the straightforward preparation of the coumarin ring. In the first step of bicoumarin synthesis, the stable and readily available tricyclic coumarin derivatives 6a, 6b and 6c were treated with PSCl3 in the presence of triethylamine at 0  C for 3 h to yield the thiophosphoryl dichloride derivatives 7aec. This step of synthesis has proven to be highly efficient for producing the thiophosphoryl dichloride derivatives 7aec as the major products, whereas the formation of di-substituted or tri-substituted thiophosphates was not observed. In the course of our research, we found that the

thiophosphoryl dichlorides 7aec were quite stable and could be isolated from reaction mixtures via column chromatography. Next, the compounds 7aec were treated with the corresponding tricyclic coumarin derivatives in a stoichiometric ratio in the presence of triethylamine to yield the bicoumarin chlorothiophosphates 8aec with good yields and a high purity level. Finally, the chlorothiophosphates 8aec were transformed into the corresponding thiophosphate bicoumarin analogs by a treatment with nucleophilic agents such as MeOH/K2CO3, H2O/K2CO3, or NH3/MeOH. The detailed synthesis of the bicoumarin thiophosphates is presented in Scheme 1.

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Scheme 1. Synthesis of the bicoumarin thiophosphates 8ae11c.

2.3. Evaluation of the STS inhibitory activities The inhibition of the STS activity was assessed using an in vitro STS assay for all of the synthesized compounds, according to the methods reported previously [28,29]. The STS enzyme was extracted from human placenta tissue and purified using a 3-step chromatography protocol. After the purification, the fractions were used directly as an enzyme source in the in vitro activity assays. Table 1 shows a summary of the results. First, we evaluated the STS inhibitory activity of the bicoumarin chlorothiophosphates 8ae8c. As expected, the compounds 8b and 8c, which have six and seven-membered hydrophobic rings within their coumarin scaffolds, exhibited a potent inhibitory effect on the STS enzyme activity with IC50 values of 7.76 and 7.04 mM, respectively. The activity of the compound 8a was slightly weaker (IC50 ¼ 33.78 mM). In addition, we found that the bicoumarin hydrogenthiophosphates 10a, 10b, and 10c exhibited the highest inhibitory effect on the STS activity, with IC50 values of 26.94, 0.86,

Table 1 Activities of the synthesized compounds and of a reference inhibitor (665COUMATE) in the STS enzyme inhibition assays. No.

Free binding energy [kcal mol1]

IC50 [mM]

8a 8b 8c 9a 9b 9c 10a 10b 10c 11a 11b 11c 665-COUMATE

5.2 6.2 7.2 4.3 4.8 6.5 6.7 8.1 6.0 6.2 5.2 2.5 5.1

33.78 7.76 7.04 75.02 14.83 6.77 26.94 0.86 5.51 74.33 109.97 88.83 1.01

± ± ± ± ± ± ± ± ± ± ± ± ±

4.6 0.6 0.7 6.8 1.6 0.7 3.4 0.07 0.5 6.0 9.4 7.3 0.3

and 5.51 mM, respectively (using an IC50 value of 1.01 mM for 665COUMATE as the reference compound). The enzyme assay results clearly showed that the hydrophobic six-membered rings in the coumarin scaffolds were the most suitable for anchoring the inhibitor within the enzyme pocket and provided the best potency. A comparison of the bicoumarin hydrogenthiophosphates 10ae10c and the bicoumarin thiophosphoroamidates 11ae11c indicated that their STS inhibitory activity was attenuated. In the case of the bicoumarin methylthiophosphates 9ae9c, lower STS inhibitory activities were also observed. The exception was the methylthiophosphate analog 9c, which showed a satisfactory activity, with an IC50 value of 6.77 mM. To explore the mode of inhibition of the bicoumarin thiophosphates, we selected the three compounds that exhibited the highest potency in STS enzyme inhibition assay: 8b, 9c and 10b (Fig. 5). As a result of this experiment, which was designed to distinguish between a reversible and an irreversible inhibition, we found that only the compound 10b allowed the enzyme to recover its activity, which suggested the reversible nature of the 10b compound. This result suggest that for the bicoumarin hydrogenthiophosphates, the hydrogen bonds established between the Thr484 residue and the hydrogenthiophosphate moieties may be critical for the stability of the enzyme-inhibitor complex and lead to a more effective inactivation of the STS. On the other hand, the compounds 8b and 9c permanently inactivated the enzyme and its activity could not be restored. Although the mechanism of action for the chlorothiophosphates is unknown, our results suggest that the nucleophilic substitution of the phosphorus atom may play a key role in the inactivation process. In this case, the bicoumarin chlorothiophosphates (e.g., 8b) may undergo a covalent binding with the OH group of the Thr484 residue, thereby providing the chemical changes in the active site of the STS enzyme. Furthermore, due to the alkylating properties of the bicoumarin methylthiophosphates (e.g., 9c), the methylation of Thr484 could not be excluded.

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Fig. 5. Reversible and irreversible inhibition of the STS enzyme by the 8b, 9c, and 10b compounds. The dotted and hatched bars refer to the basal and restored activity, respectively.

3. Conclusions A series of bicoumarin thiophosphates have been synthesized, and their affinity for the STS enzyme have been determined by means of an in vitro STS inhibition assay. Most of the new STS inhibitors possessed good activities toward the STS enzyme. We observed the most inhibitory effect of the STS enzyme using the hydrogenthiophosphate 10b compound, with an IC50 value of 860 nM (an IC50 value of 1 mM for the 665-COUMATE used as a reference). We found that the hydrogenthiophosphates (e.g., 10b) exhibited a reversible type of inhibition, whereas the chlorothiophosphates and the methylthiophosphates analogs inactivated the STS enzyme irreversibly. Docking studies conducted using these compounds have demonstrated that the new STS inhibitors exhibited a slightly different docking mode than that of the reported sulfamate-based STS inhibitor (665-COUMATE). Indeed, they can adopt conformations that are able to fill the whole cavity and prevent the substrate's access to the catalytic residues. Our studies showed that among all of the aforementioned bicoumarin derivatives, the highest level of STS inhibition was obtained with compounds that have the ability to form reversible electrostatic interactions (e.g., hydrogen bond) with the Thr484 residue. For this reason, we expect that replacing the thiophosphate moieties with a phosphate group that possesses a much stronger ability to create an electrostatic interaction (as the hydrogen bond acceptor P ¼ O) may significantly increase the inhibitory potency of the compounds.

for 13C, DMSO-d6 2.49 ppm for 1H, 39.5 ppm for 13C) or to an external standard (85% H3PO4 ¼ 0 for 31P). Coupling constants are given in Hertz. IR spectra were measured on a Nicolet 8700. CHNS elemental analysis was performed on a Carlo Erba EA-1108. Mass spectra were recorded on Agilent 6460C spectrometer. Column chromatography was performed using silica gel 60 (230e400 mesh, Merck). Preparative thin-layer chromatography was performed with Polygram SIL G/UV254 silica gel (MachereyeNagel). 4.2. General method for the synthesis of tricyclic coumarin dichlorothiophosphates 7aec To an ice-cooled solution of thiophosphoryl chloride (1.695 g, 0.1 mol) in dry THF (50 mL) was added a solution of the corresponding tricyclic coumarin (6aec) (0.1 mol) in THF dropwise, followed by triethylamine (1.01 g, 0.1 mol). The reaction mixture was stirred under a nitrogen atmosphere for 3 h. The triethylamine hydrochloride precipitate was removed by filtration, and the solvent was evaporated. The resulting residue was purified by column chromatography using CH2Cl2 as eluent to give the desired products.

4.1. Materials and methods

4.2.1. 4-oxo-1,2,3,4-tetrahydro-cyclopenta[c]chromen-7-yl dichlorothiophosphate (7a) Yield 73%, mp 68e71  C; 1H NMR dH (500 MHz, CDCl3) 2.24 (2H, quin, J 7.8, CH2), 2.93 (2H, t, J 7.8, CH2), 3.09 (2H, t, J 7.8, CH2), 7.22e7.34 (2H, m, AreH), 7.50 (1H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CDCl3) 159.3, 155.1, 154.6, 151.1 (d, JP-C 13.2), 128.3, 126.0, 117.7, 117.6 (d, JP-C 5.7), 110.2 (d, JP-C 5.7), 32.1, 30.6, 22.4; 31P NMR dP (202 MHz, CDCl3) 54.13.

Thiophosphoryl chloride and potassium carbonate are commercially available from Aldrich. Tetrahydrofuran and methanol were dried and distilled using standard procedures. Melting points (uncorrected) were determined with a Stuart Scientific SMP30 apparatus. NMR spectra were recorded on a Varian Gemini 500 MHz spectrometer. Chemical shifts are reported in ppm relative to the residual solvent peak (CDCl3 ¼ 7.26 ppm for 1H, 77.0 ppm

4.2.2. 6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl dichlorothiophosphate (7b) Yield 71%, mp 72e75  C; 1H NMR dH (500 MHz, CDCl3) 1.80e1.90 (4H, m, CH2), 2.55e2.65 (2H, m, CH2), 2.75e2.85 (2H, m, CH2), 7.21e7.30 (2H, m, AreH), 7.62 (1H, d, J 8.3, AreH); 13C NMR dC (125 MHz, CDCl3) 161.3, 152.7, 150.9 (d, JP-C 13.2), 146.6, 124.8 (d, JP-C 2.2), 124.5, 119.4, 117.6 (d, JP-C 5.3), 110.3 (d, JP-C 6.1), 25.5, 24.3, 21.6,

4. Experimental

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21.4;

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P NMR dP (202 MHz, CDCl3) 54.15.

4.2.3. 6-oxo-6,7,8,9,10,11-hexahydro-cyclohepta[c]chromen-3-yl dichlorothiophosphate (7c) Yield 70%, mp 108e110  C; 1H NMR dH (500 MHz, CDCl3) 1.59e1.73 (4H, m, CH2), 1.89e1.95 (2H, m, CH2), 2.90e2.98 (4H, m, CH2), 7.22e7.31 (2H, m, AreH), 7.72e7.75 (1H, m, AreH); 13C NMR dC (125 MHz, CDCl3) 161.6, 153.3, 153.1, 151.1 (d, JP-C 13.2), 129.4, 125.7 (d, JP-C 2.2), 119.1, 117.6 (d, JP-C 5.7), 110.4 (d, JP-C 6.1), 32.1, 28.5, 27.1, 25.7, 25.1; 31P NMR dP (202 MHz, CDCl3) 53.99. 4.3. General method for the synthesis of bicoumarin chlorothiophosphates 8ae8c To a solution of tricyclic coumarin dichlorothiophosphates (7aec) (1.0 mmol) in dry THF (15 mL) was added the corresponding solution of tricyclic coumarin (6aec) (1.0 mmol) followed by triethylamine (1 mmol). The reaction mixture was stirred under a nitrogen atmosphere for 24 h. The triethylamine hydrochloride precipitate was removed by filtration, and the solvent was evaporated. The resulting residue was purified by column chromatography using CHCl3: AcOEt 15:1 as eluent to give the desired products. 4.3.1. Bis-(4-oxo-1,2,3,4-tetrahydro-cyclopenta[c]chromen-7-yl) chlorothiophosphate (8a) Yield 48%, mp 178e181  C; vmax (KBr)/cm1 1717, 1603, 1568, 1498, 1427, 1383, 1267, 1127, 1015, 975, 900, 751, 695; 1H NMR dH (500 MHz, CDCl3) 2.24 (4H, quin, J 7.6, CH2), 2.92 (4H, t, J 7.3, CH2), 3.09 (4H, t, J 7.6, CH2), 7.24e7.33 (4H, m, AreH), 7.50 (2H, d, J 8.3, AreH); 13C NMR dC (125 MHz, CDCl3) 159.9, 155.8, 154.8, 151.3 (d, JPC 9.7), 128.3, 126.3, 117.8 (d, JP-C 5.3), 117.7, 110.2 (d, JP-C 5.3), 32.4, 30.9, 22.7; 31P NMR dP (202 MHz, CDCl3) 58.36. Anal. Calcd for: C24H18ClO6PS: C, 57.55; H, 3.62; S, 6.40. Found: C, 57.64; H, 3.69; S, 6.47; () ESI MS molecular ion (m/z): 499.0, fragmentation ions: 930.8, 480.9, 464.9, 200.9. 4.3.2. Bis-(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl) chlorothiophosphate (8b) Yield 55%, mp 163e168  C; vmax (KBr)/cm1 1713, 1605, 1575, 1500, 1425, 1384, 1258, 1129, 1020, 972, 906, 750, 695; 1H NMR dH (500 MHz, CDCl3) 1.80e1.89 (8H, m, CH2), 2.56e2.62 (4H, m, CH2), 2.75e2.80 (4H, m, CH2), 7.22e7.28 (4H, m, AreH), 7.61 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CDCl3) 161.7, 152.7, 150.9 (d, JP-C 9.7), 147.1, 124.9, 124.2, 119.1, 117.6 (d, JP-C 4.8), 110.1 (d, JP-C 5.3), 25.6, 24.2, 21.6, 21.4; 31P NMR dP (202 MHz, CDCl3) 58.42. Anal. Calcd for: C26H22ClO6PS: C, 59.04; H, 4.19; S, 6.06. Found: C, 59.15; H, 4.25; S, 6.11; () ESI MS molecular ion (m/z): 527.0, fragmentation ions: 987.7, 508.9, 493.0. 4.3.3. Bis-(6-oxo-6,7,8,9,10,11-hexahydro-cyclohepta[c]chromen-3yl) chlorothiophosphate (8c) Yield 43%, mp 185e189  C; vmax (KBr)/cm1 1712, 1606, 1569, 1504, 1420, 1378, 1263, 1124, 1005, 973, 900, 757, 693; 1H NMR dH (500 MHz, CDCl3) 1.61e1.92 (12H, m, CH2), 2.91e2.98 (8H, m, CH2), 7.24e7.31 (4H, m, AreH), 7.73 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CDCl3) 161.7, 153.4, 153.2, 151.1 (d, JP-C 9.7), 129.1, 125.7, 118.8, 117.5 (d, JP-C 4.8), 110.2, 32.2, 28.5, 27.1, 25.8, 25.1; 31P NMR dP (202 MHz, CDCl3) 58.16. Anal. Calcd for: C28H26ClO6PS: C, 60.38; H, 4.70; S, 5.76. Found: C, 60.33; H, 4.74; S, 5.83; () ESI MS molecular ion (m/z): 555.1, fragmentation ions: 536.0, 521.0, 448.7, 305.9, 254.8, 229.0, 212.7, 168.9.

4.4. General method for the synthesis of bicoumarin methylthiophosphates 9ae9c To a solution of corresponding bicoumarin chlorothiophosphates (8ae8c) (1.0 mmol) in dry THF (10 mL) was added methanol (1 mmol) followed by potassium carbonate (1 mmol). The reaction mixture was stirred under a nitrogen atmosphere for 24 h and filtered. After concentration under vacuum, the resulting residue was purified by column chromatography using CHCl3: AcOEt 10:1 as eluent to give the desired products. 4.4.1. Bis-(4-oxo-1,2,3,4-tetrahydro-cyclopenta[c]chromen-7-yl) methylthiophosphate (9a) Yield 62%, mp 175e179  C; vmax (KBr)/cm1 1714, 1606, 1569, 1506, 1459, 1425, 1388, 1330, 1275, 1130, 1028, 980, 896, 845, 776, 586; 1H NMR dH (500 MHz, CDCl3) 2.20e2.26 (4H, m, CH2), 2.90e3.10 (8H, m, CH2), 4.00 (3H, d, J 14.2, CH3), 7.15e7.23 (4H, m, AreH), 7.45 (2H, d, J 7.8, AreH); 13C NMR dC (125 MHz, CDCl3) 159.9, 155.8, 154.9, 152.1, 127.8, 126.1, 117.6, 116.9, 109.8 (d, JP-C 4.8), 56.5, 32.4, 30.8, 22.7; 31P NMR dP (202 MHz, CDCl3) 60.13. Anal. Calcd for: C25H21O7PS: C, 60.48; H, 4.26; S, 6.46. Found: C, 60.59; H, 4.34; S, 6.51; () ESI MS molecular ion (m/z): 495.1, fragmentation ions: 480.9, 464.9, 201.0. 4.4.2. Bis-(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl) methylthiophosphate (9b) Yield 60%, mp 184e188  C; vmax (KBr)/cm1 1699, 1606, 1573, 1501, 1461, 1425, 1385, 1330, 1279, 1140, 1035, 973, 905, 850, 770, 585; 1H NMR dH (500 MHz, CDCl3) 1.80e1.87 (8H, m, CH2), 2.55e2.80 (8H, m, CH2), 4.00 (3H, d, J 14.2, CH3), 7.13e7.17 (4H, m, AreH), 7.56 (2H, d, J 8.3, AreH); 13C NMR dC (125 MHz, CDCl3) 161.6, 152.8, 151.6 (d, JP-C 7.5), 146.9, 124.7, 123.6, 118.3, 117.4 (d, JP-C 4.8), 109.6 (d, JP-C 4.8), 56.4 (d, JP-C 5.7), 25.5, 24.2, 21.7, 21.5; 31P NMR dP (202 MHz, CDCl3) 60.18. Anal. Calcd for: C27H25O7PS: C, 61.83; H, 4.80; S, 6.11. Found: C, 61.95; H, 4.87; S, 6.19; () ESI MS molecular ion (m/z): 523.1, fragmentation ions: 536.0, 508.9, 492.9, 214.9. 4.4.3. Bis-(6-oxo-6,7,8,9,10,11-hexahydro-cyclohepta[c]chromen-3yl) methylthiophosphate (9c) Yield 55%, mp 96e102  C; vmax (KBr)/cm1 1714, 1606, 1569, 1504, 1455, 1422, 1378, 1328, 1276, 1129, 1027, 974, 891, 852, 774, 583; 1H NMR dH (500 MHz, CDCl3) 1.59e1.71 (8H, m, CH2), 1.88e1.93 (4H, m, CH2), 2.88e2.96 (8H, m, CH2), 4.00 (3H, d, J 14.2, CH3), 7.14e7.20 (4H, m, AreH), 7.67 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CDCl3) 161.9, 153.4, 151.8 (d, JP-C 7.5), 128.5, 125.5, 118.0, 117.4, 109.8, 56.4, 32.2, 28.5, 27.0, 25.8, 25.1; 31P NMR dP (202 MHz, CDCl3) 59.96. Anal. Calcd for: C29H29O7PS: C, 63.03; H, 5.29; S, 5.80. Found: C, 63.15; H, 5.37; S, 5.89; () ESI MS molecular ion (m/z): 551.1, fragmentation ions: 536.9, 520.9, 309.0, 229.0, 214.9. 4.5. General method for the synthesis of bicoumarin hydrogenthiophosphates 10ae10c To a solution of corresponding bicoumarin chlorothiophosphates (8ae8c) (1.0 mmol) in THF (10 mL) was added a water solution of potassium carbonate (0.5 mL). The reaction mixture was stirred for 24 h. After concentration under vacuum, the resulting residue was purified by column chromatography using AcOEt:MeOH 20:1 as eluent to give the desired products. 4.5.1. Bis-(4-oxo-1,2,3,4-tetrahydro-cyclopenta[c]chromen-7-yl) hydrogenthiophosphate (10a) Yield 34%, mp 166  C (with decomposition); vmax (KBr)/cm1 3410, 1693, 1605, 1563, 1506, 1428, 1392, 1252, 1129, 973, 854, 728; 1 H NMR dH (500 MHz, CD3OD) 2.20 (4H, quin, J 7.6, CH2), 2.81 (4H, t,

S. Demkowicz et al. / European Journal of Medicinal Chemistry 101 (2015) 358e366

J 7.3, CH2), 3.07 (4H, t, J 7.3, CH2), 7.22e7.32 (4H, m, AreH), 7.46 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CD3OD) 161.0, 157.4, 155.1, 154.7, 125.6, 125.4, 118.0 (d, JP-C 5.3), 114.8, 108.7 (d, JP-C 5.3), 31.8, 30.1, 22.3; 31P NMR dP (202 MHz, CD3OD) 49.03. Anal. Calcd for: C24H19O7PS: C, 59.75; H, 3.97; S, 6.65. Found: C, 59.69; H, 4.03; S, 6.71; () ESI MS molecular ion (m/z): 481.1, fragmentation ions: 510.9, 496.9, 480.9, 464.9, 220.9. 4.5.2. Bis-(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl) hydrogenthiophosphate (10b) Yield 45%, mp 187  C (with decomposition); vmax (KBr)/cm1 3418, 1695, 1606, 1568, 1504, 1426, 1389, 1259, 1135, 968, 852, 724; 1 H NMR dH (500 MHz, DMSO) 1.70e1.76 (8H, m, CH2), 2.38e2.50 (4H, m, CH2), 2.69e2.80 (4H, m, CH2), 3.8e4.3 (1H, brs, OH) 7.16e7.21 (4H, m, AreH), 7.61 (2H, d, J 8.3, AreH); 13C NMR dC (125 MHz, DMSO) 161.4, 155.2 (d, JP-C 7.5), 152.6, 148.0, 125.1, 121.2, 117.8 (d, JP-C 5.3), 115.8, 108.3 (d, JP-C 5.3), 25.3, 24.3, 21.8, 21.5; 31P NMR dP (202 MHz, DMSO) 46.00. Anal. Calcd for: C26H23O7PS: C, 61.17; H, 4.54; S, 6.28. Found: C, 61.29; H, 4.61; S, 6.38; () ESI MS molecular ion (m/z): 509.1, fragmentation ions: 524.9, 493.0, 215.0. 4.5.3. Bis-(6-oxo-6,7,8,9,10,11-hexahydro-cyclohepta[c]chromen-3yl) hydrogenthiophosphate (10c) Yield 37%, mp 209  C (with decomposition); vmax (KBr)/cm1 3411, 1685, 1603, 1560, 1504, 1425, 1381, 1266, 1135, 975, 857, 724; 1 H NMR dH (500 MHz, CD3OD) 1.51e1.61 (8H, m, CH2), 1.84e1.90 (4H, m, CH2), 2.79e2.93 (8H, m, CH2), 7.22e7.25 (4H, m, AreH), 7.67 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, CD3OD) 162.8, 155.2, 154.9 (d, JP-C 7.9), 153.1, 126.2, 125.2, 117.9 (d, JP-C 5.3), 115.8, 108.7 (d, JP-C 5.3), 31.9, 27.8, 26.3, 25.6, 25.0; 31P NMR dP (202 MHz, CD3OD) 48.50. Anal. Calcd for: C28H27O7PS: C, 62.45; H, 5.05; S, 5.95. Found: C, 62.37; H, 5.11; S, 6.03; () ESI MS molecular ion (m/z): 537.1. 4.6. General method for the synthesis of bicoumarin thiophosphoroamidates 11ae11c To a solution of corresponding bicoumarin chlorothiophosphates (8ae8c) (1.0 mmol) in THF (10 mL) was added a methanol solution of NH3 (1 mL). The reaction mixture was stirred for 1 h. After concentration under vacuum, the resulting residue was purified by column chromatography using CHCl3:AcOEt 15:1 as eluent to give the desired products. 4.6.1. Bis-(4-oxo-1,2,3,4-tetrahydro-cyclopenta[c]chromen-7-yl) thiophosphoroamidate (11a) Yield 58%, mp 212e214  C; vmax (KBr)/cm1 3279, 1708, 1606, 1550, 1507, 1429, 1388, 1267, 1126, 968, 875, 819, 750, 588; 1H NMR dH (500 MHz, DMSO) 2.10 (4H, quin, J 7.3, CH2), 2.74 (4H, t, J 7.1, CH2), 3.06 (4H, t, J 7.1, CH2), 6.45 (2H, d, JP-N 7.3, NH2), 7.27e7.36 (4H, m, AreH), 7.63 (2H, d, J 8.3, AreH); 13C NMR dC (125 MHz, DMSO) 159.5, 156.6, 154.7, 152.9 (d, JP-C 7.0), 127.2, 126.7, 118.5, 116.2, 109.7, 32.4, 30.9, 22.6; 31P NMR dP (202 MHz, DMSO) 66.86. Anal. Calcd for: C24H20NO6PS: C, 59.87; H, 4.19; N, 2.91; S, 6.66. Found: C, 59.79; H, 4.12; N, 2.99; S, 6.72; () ESI MS molecular ion (m/z): 480.1, fragmentation ions: 277.9, 201.0, 168.8. 4.6.2. Bis-(6-oxo-7,8,9,10-tetrahydro-6H-benzo[c]chromen-3-yl) thiophosphoroamidate (11b) Yield 60%, mp 205e209  C; vmax (KBr)/cm1 3311, 1700, 1606, 1541, 1500, 1426, 1385, 1259, 1134, 967, 885, 810, 756, 582; 1H NMR dH (500 MHz, DMSO) 1.68e1.75 (8H, m, CH2), 2.36e2.42 (4H, m, CH2), 2.71e2.80 (4H, m, CH2), 6.44 (2H, d, JP-N 7.8, NH2), 7.24e7.28 (4H, m, AreH), 7.70 (2H, d, J 8.8, AreH); 13C NMR dC (125 MHz, DMSO) 161.0, 152.5, 151.4 (d, JP-C 6.6), 147.7, 125.8, 122.5, 118.2, 117.6, 109.4, 25.4, 24.3, 21.7, 21.4; 31P NMR dP (202 MHz, DMSO) 66.83.

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Anal. Calcd for: C26H24NO6PS: C, 61.29; H, 4.75; N, 2.75; S, 6.29. Found: C, 61.36; H, 4.82; N, 2.86; S, 6.24. () ESI MS molecular ion (m/z): 508.1, fragmentation ions: 535.9, 291.9, 254.9, 228.9, 214.9, 186.9, 168.8. 4.6.3. Bis-(6-oxo-6,7,8,9,10,11-hexahydro-cyclohepta[c]chromen-3yl) thiophosphoroamidate (11c) Yield 52%, mp 240e244  C; vmax (KBr)/cm1 3251, 1684, 1604, 1557, 1500, 1423, 1376, 1265, 1134, 972, 879, 820, 746, 584; 1H NMR dH (500 MHz, DMSO) 1.48e1.60 (8H, m, CH2), 1.80e1.90 (4H, m, CH2), 2.76e3.00 (8H, m, CH2), 6.45 (2H, d, JP-N 6.8, NH2), 7.26e7.31 (4H, m, AreH), 7.95 (2H, d, J 8.3, AreH); 13C NMR dC (125 MHz, DMSO) 161.5, 154.2, 153.2, 152.7 (d, JP-C 6.6), 127.6, 126.7, 118.3 (d, JP31 C 4.8), 117.4, 109.7 (d, JP-C 5.3), 32.1, 28.1, 26.8, 25.9, 25.2; P NMR dP (202 MHz, DMSO) 66.76. Anal. Calcd for: C28H28NO6PS: C, 62.56; H, 5.25; N, 2.61; S, 5.96. Found: C, 62.67; H, 5.33; N, 2.72; S, 5.87. (-) ESI MS molecular ion (m/z): 536.1, fragmentation ions: 520.9, 493.0, 480.9, 464.9, 325.1, 284.9229.0, 201.0. 4.7. Biological assays 4.7.1. Enzyme purification STS was extracted from human placenta and purified to homogeneity following a multi-step chromatography protocol as previously described [30]. 4.7.2. In vitro activity assay The reaction mixture, at a final volume of 100 ml, contained 20 mM TriseHCl pH 7.4, 3 mM NPS, various concentrations of inhibitor (0.1e200 mM) and 5 U of purified enzyme (1 U is the amount of enzyme that hydrolyzes 100 mM of NPS in 1 h at 37  C). The reaction was performed at 37  C for 15 min. It was halted by the addition of 100 ml of 1 M NaOH. The absorbance of the released pnitrophenol was measured at 405 nm using a Microplate Reader Biotek ELx800 (SERVA). IC50 values were calculated using GraphPad Prism software. All measurements were performed in triplicate. To discriminate between reversible and irreversible inhibition of STS by the inhibitors, standard assays in presence of 100 mM of compound 8b, 9c and 10b were performed. Reaction mixture was scale up to 200 ml of total volume and was conducted in standard conditions for 30 min. Next, the reaction mixture was split into two parts. One part was stopped immediately (reference) and the second part was transferred to centricon tube (MWCO 10 kDa) in order to completely exchange the reaction environment. Finally the restored enzyme activity was measured as described above. Acknowledgements We gratefully acknowledge the National Science Centre (Poland) for financial support (grant no. 2011/03/D/NZ7/03985). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.06.051. References [1] C.R. Ireson, S.K. Chander, A. Purohit, D.C. Parish, L.W.L. Woo, B.V.L. Potter, M.J. Reed, Pharmacokinetics of the nosteroidal steroid sulphatase inhibitor 667 COUMATE and its sequestration into red blood cells in rats, Br. J. Cancer 91 (2004) 1399e1404.
ski, [2] S. Demkowicz, W. Kozak, M. Da
sko, A. Wołos, M. Masłyk, K. Kubin A. Składanowski, M. Misiak, J. Rachon, Synthesis and steroid sulfatase inhibitory activities of N-alkanoyl tyramine phosphates and thiophosphates, RSC Adv. 5 (2015) 32594e32603. [3] M.J. Reed, A. Purohit, L.W.L. Woo, S.P. Newman, B.V.L. Potter, Steroid sulfatase:

366

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

S. Demkowicz et al. / European Journal of Medicinal Chemistry 101 (2015) 358e366 molecular biology, regulation, and inhibition, Endocr. Rev. 26 (2005) 171e202. F.G. Hernandez-Guzman, T. Higashiyama, W. Pangborn, Y. Osawa, D. Ghosh, Structure of human estrone sulfatase suggests functional roles of membrane association, J. Biol. Chem. 278 (2003) 22989e22997. T. Selmer, A. Hallmann, B. Schmidt, M. Sumper, K. von Figura, The evolutionary conservation of a novel protein modification, the conversion of cysteine to serine-semialdehyde in arylsulfatase from Volvox carteri, Eur. J. Biochem. 238 (1996) 341e345. M. Recksiek, T. Selmer, T. Dierks, B. Schmidt, K. von Figura, Sulfatases, trapping of the sulphated enzyme intermediate by substituting the active site formylglycine, J. Biol. Chem. 273 (1998) 6096e6103. A. Purohit, M.J. Reed, N.C. Morris, G.J. Williams, B.V.L. Potter, Regulation and inhibition of steroid sulfatase activity in breast cancer, Ann. N. Y. Acad. Sci. 784 (1996) 40e49. W. Elger, S. Schwarz, A. Hedden, G. Reddersen, B. Schneider, Sulfamates of various estrogens are prodrugs with increased systemic and reduced hepatic estrogenicity at oral application, J. Steroid Biochem.. Mol. Biol. 55 (1995) 395e403. A. Purohit, L.W.L. Woo, B.V.L. Potter, M.J. Reed, In vivo inhibition of estrone sulfatase activity and growth of nitrosomethylurea-induced mammary tumors by 667 COUMATE, Cancer Res. 60 (2000) 3394e3396. P.A. Foster, S.P. Newman, S.K. Chander, C. Stengel, R. Jhalli, L.L.W. Woo, In vivo efficacy of STX213, a second-generation steroid sulfatase inhibitor, for hormone-dependent breast cancer therapy, Clin. Cancer Res. 12 (2006) 5543e5549. P. Foster, S. Chander, M. Parsons, S. Newman, L. Woo, B. Potter, Efficacy of three potent steroid sulfatase inhibitors: pre-clinical investigations for their use in the treatment of hormone-dependent breast cancer, Breast Cancer Res. Treat. 111 (2008) 129e138. H. Ishida, T. Nakata, M. Suzuki, Y. Shiotsu, H. Tanaka, N. Sato, A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models, Breast Cancer Res. Treat. 106 (2007) 215e227. H. Ishida, T. Nakata, N. Sato, P. Li, T. Kuwabara, S. Akinaga, Inhibition of steroid sulfatase activity and cell proliferation in ZR-75-1 and BT-474 human breast cancer cells by KW-2581 in vitro and in vivo, Breast Cancer Res. Treat. 104 (2007) 211e219. Y. Yang, Q.W. Liu, Y. Shi, Z.G. Song, Y.H. Jin, Z.Q. Liu, Design and synthesis of coumarin-3-acylamino derivatives to scavenge radicals and to protect DNA, Eur. J. Med. Chem. 84 (2014) 1e7.
nchez-Recillas, G. Navarrete-Va
zquez, S. Hidalgo-Figueroa, M. Yolanda A. Sa Rios, M. Ibarra-Barajas, S. Estrada-Soto, Semisynthesis, ex vivo evaluation, and SAR studies of coumarin derivatives as potential antiasthmatic drugs, Eur. J. Med. Chem. 77 (2014) 400e408.

[16] M.I. El-Gamal, ChH. Oh, Synthesis, in vitro antiproliferative activity, and in silico studies of fused tricyclic coumarin sulfonate derivatives, Eur. J. Med. Chem. 84 (2014) 68e76. [17] R. Pingaew, A. Saekee, P. Mandi, Ch Nantasenamat, S. Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, Synthesis, biological evaluation and molecular docking of novel chalcone-coumarin hybrids as anticancer and antimalarial agents, Eur. J. Med. Chem. 85 (2014) 65e76. [18] H. Hussain, J. Hussain, A. Al-Harrasi, K. Krohn, The chemistry and biology of bicoumarins, Tetrahedron 68 (2012) 2553e2578. [19] M.S. Frasinyuk, S.P. Bondarenko, V.P. Khilya, Chemistry of 3-hetarylcoumarins. 3. Synthesis and aminomethylation of 70 -hydroxy-3,40 -bicoumarins, Chem. Heterocycl. Compd 48 (2012) 422e426. [20] E. Ziegler, H. Junek, H. Kroboth, Syntheses of heterocycles, 188. On the chemistry of meldrum acid, III, Monatsh. Chem. 107 (1976) 317e324. [21] A. Chandrasekhar, P. Padmanabhan, S. Seshadri, Synthesis of biscoumarins and biscoumarinyl ketones, Dyes Pigment 7 (1986) 13e21. [22] M. llyas, M. Parveen, Biomimetic synthesis of some novel coumarin dimers, Tetrahedron 52 (1996) 3991e3996. [23] G. Buchi, D.H. Klaubrt, R.C. Schank, S.M. Weinred, G.N. Wogan, Structure and synthesis of kotanin and desmethylkotanin, metabolites of Aspergillus glaucus, J. Org. Chem. 36 (1971) 1143e1147. [24] B.H. Lipschutz, E. Kayser, Z.P. Liu, Asymmetric synthesis of biaryls by intramolecular oxidative couplings of cyanocuprate intermediates, Angew. Chem. Int. Ed. Engl. 33 (1994) 1842e1844. [25] W. Huettel, M. Nieger, M. Mueller, A short and efficient total synthesis of the naturally occurring coumarins siderin, kotanin, isokotanin A and desertorin C, Synthesis 12 (2003) 1803e1808. [26] G. Bringmann, J. Hinrichs, P. Henschel, J. Kraus, K. Peters, E.M. Peters, Novel concepts in directed biaryl synthesis, 97. Atropo-enantioselective synthesis of the natural bicoumarin (þ)-isokotanin A via a configurationally stable biaryl lactone, Eur. J. Org. Chem. 6 (2002) 1096e1106. [27] W. Kozak, M. Da
sko, M. Masłyk, J.S. Pieczykolan, B. Gielniewski, J. Rachon, S. Demkowicz, Phosphate tricyclic coumarin analogs as steroid sulfatase inhibitors: synthesis and biological activity, RSC Adv. 4 (2014) 44350e44358. [28] A.M. Vaccaro, R. Salvioli, M. Muscillo, L. Renola, Purification and properties of arylsulfatase C from human placenta, Enzyme 37 (1987) 115e126. [29] L.W.L. Woo, T. Jackson, A. Putey, G. Cozier, P. Leonard, K.R. Acharya, S.K. Chander, A. Purohit, M.J. Reed, B.V.L. Potter, Highly potent first examples of dual aromatase-steroid sulfatase inhibitors based on a biphenyl template, J. Med. Chem. 53 (2010) 2155e2170. [30] F.G. Hernandez-Guzman, T. Higashiyama, Y. Osawa, D. Ghosh, Purification, characterization and crystallization of human placental estrone/dehydroepiandrosterone sulfatase, a membrane-bound enzyme of the endoplasmic reticulum, J. Steroid Biochem. Mol. Biol. 78 (2001) 441e450.