Design, Synthesis, and Biological Evaluation of a Series of 5- and 7-Hydroxycoumarin Derivatives as 5-HT_1A Serotonin Receptor Antagonists

Abstract

We have designed and synthesized a series of 60 new 5- and 7-hydroxycoumarin derivatives bearing the piperazine moiety with the expected binding to 5-HT_1A and 5-HT_2A receptors. Molecular docking of all investigated compounds revealed subnanomolar estimates of 5-HT_1AR K_i for three ligands and 5-HT_2AR Ki for one ligand as well as numerous low nanomolar estimates of K_i for both receptors. Intrigued by these results we synthesized all 60 new derivatives using microwave-assisted protocols. We show that three new compounds show a relatively high antagonistic activity against the 5HT_1A receptor, although lower than the reference compound WAY-100635. These compounds also showed relatively low binding affinities to the 5-HT_2A receptor. We also provide a detailed structure–activity analysis of this series of compounds and compare it with previously obtained results for an exhaustive series of coumarin derivatives.

1. Introduction

N-arylpiperazine-containing ligands are a large class of chemical compounds with various known biological activities, such as enzyme inhibition, antibacterial, antineoplastic, and anticancer properties, as well as adrenergic and serotonin receptor inhibition [1,2,3,4,5,6,7]. This last activity is particularly prominent for this family of compounds, as even some of its simplest members, such as 1-(3-chlorophenyl) piperazine or m-trifluoromethylphenylpiperazine, are known to interact with serotonin receptors [4,8]. The high affinity of these systems to 5HT receptors stems from the highly basic nitrogen atom of the piperazine, which is able to form strong interactions with the conserved acidic amino acids in the GPCR transmembrane domain of these proteins [9]. In order to be effective as 5HT receptor antagonists or agonists, such compounds require in their structure, however, also a relatively bulky moiety connected usually to the N-arylpiperazine via a flexible aliphatic linker. Such a design principle has been first realized in buspirone, a 5-HT_1A receptor agonist, which also has moderate activity against other 5HT receptors and selected dopamine receptors, and is followed until today with the goal of finding new agonists/antagonists of 5HT receptors [10,11,12]. 5HT receptors proteins modulate the release of many neurotransmitters, therefore are an important target for a variety of drugs, including antipsychotics, antidepressants, hallucinogens anorectics, and antimigraine agents [13,14,15,16].

Among many groups considered as the bulky moiety connected to N-arylpiperazine coumarin derivatives have gained some attention, particularly after the investigations of Chen et al., who showed that selected N-arylpiperazines connected to coumarins in position 7 via a (CH₂)₄ linker have nanomolar Ki values toward 5-HT_1A and 5-HT_2A receptors [17,18]. Inspired by these works we have expanded the family of potential serotonin agonists/antagonists based on the same design principle by introducing different arylpiperazine derivatives of 7-hydroxycoumarin, some of which showed subnanomolar affinities to 5-HT_1A receptor and low nanomolar affinities to 5-HT_2A receptor [19,20]. Later we have also obtained a series of arylpiperazine derivatives of 5-hydroxycoumarin [21,22,23]. We showed that the highest, subnanomolar affinities for 5-HT_1A receptor were associated with the presence of the acetyl group in the C-6 position at the coumarin ring and the substituents in the 2 or 3 position in the phenyl ring of piperazine. Finally, in 2020 we designed a new series of arylpiperazinyl derivatives of 6-acetyl-5-hydroxy-4,7-dimethylcoumarin, which also showed very low nanomolar affinities toward 5HT_1A and 5HT_2A but also low affinities to the D₂ receptor [24]. In these studies we noticed that the length of the alkyl linker (three-carbon versus four-carbon) had little impact on the obtained Ki values, since the affinities for specific serotonin receptors for analogous compounds containing the same arylpiperazinyl fragments, differing only in the length of the alkyl linker, were very similar. It is worth noting that this finding is not based on single cases but on a large number of cases, showing a clear tendency for this particular length of the linker (Figure 1).

To conclude our search for new biologically active compounds in this series as well as to gain even more knowledge of the structure–activity relationships we have designed two new series of arylpiperazine derivatives of 5-hydroxycoumarins and 7-hydroxycoumarins. These series were designed based on the aryl substituents giving the highest affinities in our previous studies, but with different lengths of the alkyl linkers, consisting of either two or five CH₂ moieties. In this study we have used molecular docking to homology models of 5-HT_1A and 5-HT_2A receptors followed by microwave-assisted protocols to synthesize all 60 compounds. We also performed functional activity studies for the 5-HT_1A receptor, as well as 5-HT_2A receptor affinity studies.

2. Results and Discussion

2.1. Docking Studies

The results of the Ki estimates obtained from the computational studies are presented in

Table 1. Computational Ki values for compounds for compounds of series 1, 2, and 3.

Compound	5-HT1A Ki[nM]	5-HT2A Ki[nM]
1	206.3	1023
1a	0.4	9.64
1b	3.2	36.1
1c	1.2	7.7
1d	2.6	19.6
1e	5.0	9.2
1f	10.8	30.5
1g	4.6	6.4
1h	1.6	2.4
1i	4.0	2.0
1j	10.0	1.1
2	922.3	6610
2a	3.9	22.3
2b	6.6	44.8
2c	8.0	29.8
2d	1.5	12.7
2e	13.1	40.3
2f	31.9	12.4
2g	4.6	4.4
2h	9.4	20.6
2i	5.9	11.3
2j	16.6	18.9
3	154.4	3080
3a	35.5	33.6
3b	23.3	53.2
3c	5.9	6.8
3d	928.5	7.0
3e	106.2	59.7
3f	1.9	1.1
3g	3.2	10.0
3h	12.4	11.8
3i	3.6	5.2
3j	37.2	31.5
ketanserin	71.3	58.7
WAY-100635	50.5	73.9

and

Table 2. Computational Ki values for compounds for compounds of series 4, 5, and 6.

Compound	5-HT1A Ki[nm]	5-HT2A Ki[nm]
4	429.2	3270
4a	1.4	20.8
4b	3.1	44.7
4c	10.3	16.3
4d	10.8	30.8
4e	52.2	17.1
4f	17.7	25.5
4g	3.9	1.6
4h	7.3	17.8
4i	2.0	5.1
4j	2.7	2.1
5	57.7	2100
5a	3.5	16.1
5b	25.7	27.2
5c	72.2	5.8
5d	8.3	2.7
5e	12.5	3.5
5f	58.5	5.2
5g	4.7	2.9
5h	20.6	5.0
5i	20.4	5.2
5j	3.7	0.9
6	171.6	4430
6a	17.3	10.8
6b	0.7	25.4
6c	1.9	8.7
6d	2.6	5.8
6e	3.7	36.9
6f	4.6	6.9
6g	0.3	10.3
6h	1.5	9.9
6i	2.8	9.9
6j	10.0	2.9
ketanserin	71.3	58.7
WAY-100635	50.5	73.9

. While for the starting compounds 1–6 the Ki values were estimated at 56–922 nM for 5-HT_1A and above 1 μm for 5-HT_2A receptor, some of the functionalized derivatives show nanomolar or even subnanomolar affinities. In particular, there are three new compounds with the estimated Ki versus 5-HT_1A receptor below 1 nm (1a, 6b, and 6g) and one for 5-HT_2A receptor (5j). As there are many other compounds with the estimated Ki below 10 nM we decided to synthesize all of these systems to verify their 5-HT_1A/2A receptor affinities. We also decided to perform a detailed analysis of the predicted binding poses for 5-HT_1A receptor, as the Ki values for the 5HT_2A receptor are usually less favorable and our previous studies showed that this class of compounds in most cases binds stronger to 5-HT_1A receptor than to 5-HT_2A receptor [19,20,21,22,23,24].

Of the three compounds predicted to have subnanomolar affinities to 5-HT_1A receptor (1a, 6b, 6g) all have the crucial interaction between the basic nitrogen atom of the piperazine group with the conserved D116 of the receptor binding site (see Figure 2). As one can see the poses of these three systems are also very similar, with the coumarin part going deep into the binding pocket, toward transmembrane (TM) helix 7 and the arylpiperazine extending toward transmembrane helix 4. Apart from the salt bridge to D114 from transmembrane helix 3, 1a is predicted to form also hydrogen bonds between the methoxy moiety of the arylpiperazine and S199 (TM4) as well as between the coumarin part and N392 (TM7). On the other hand 6b is predicted to be additionally stabilized by the interaction between the F atom of the arylpiperazine and S199 (TM4), while 6g by the hydrogen bond between the oxygen atom of the linker and Y390 (TM7). It is also worth mentioning that these poses are similar to our previously predicted poses for the coumarin derivatives with three CH₂ moieties. On the other hand for some derivatives with four CH₂ groups we predicted a different orientation of the ligand, where the coumarin part extends toward TM helix 4, while the arylpiperazine part goes deep into the pocket and interacts with the residues located on TM7 [24].

2.2. Chemistry

The starting coumarins 5-hydroxy-4,7-dimethylchromen-2-one (A), 6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one (B), and 8-acetyl-7-hydroxy-4-methylchromen-2-one (C) were resynthesized according to previously published studies [25,26]. The reaction of starting coumarins (A–C) with 1,2-dibromoethane in acetonitrile in the presence of potassium iodide and potassium carbonate afforded with different yields (25–80%) 5-(2-bromo ethoxy)-4,7-dimethyl-2H-chromen-2-one (2), 6-acetyl-5-(2-bromoethoxy)-4,7-dimethyl-2H-chromen-2-one (4) and 8-acetyl-7-(2-bromoethoxy)-4-methylchromen-2-one (6), while upon reaction of 1,5-dibromopentane, in the same conditions, 5-(5-bromopentyloxy)-4,7-dimethyl-2H-chromen-2-one (1), 6-acetyl-5-(5-bromopentyloxy)-4,7-dimethyl-2H-chromen-2-one (3) and 8-acetyl-7-(5-brompenthoxy)-4-methylchromen-2-one (5) were obtained with different yields (44–89%). In the next step, the final compounds were synthesized as pictured in Scheme 1 and according to the previously published study [19]. The synthesis of compounds 1a–1j, 2a–2j, 3a–3j, 4a–4j, 5a–5j, and 6a–6j was carried out by reacting the bromoalkyl derivatives (1–6) with appropriate arylpiperazine: (4-(2-methoxyphenyl)piperazine, (4-(2-fluorophenyl) piperazine, (4-(3-methoxyphenyl)piperazine, (4-(2,5-dimethylyphenyl)piperazine, (4-(3-fluorophenyl)piperazine, (4-(2-bromophenyl)piperazine, (4-(3-bromophenyl) piperazine, (4-(3,5-dimethylphenyl)piperazine, (4-(2,3-dichlorophenyl)piperazine, (4-(2-cyano phenyl)piperazine) in acetonitrile and in the presence of potassium iodide and potassium carbonate. Reaction progress was monitored by TLC using silica gel plates (eluent: CHCl3: MeOH; 10:0.25). All compounds synthesized in this work were obtained using a microwave reactor and were purified by column chromatography using silica gel and CHCl₃:MeOH (100:1) as the eluent, as in the previously published study [19]. All syntheses were performed in the millimolar scale, starting from 1 mmol of the starting coumarin derivatives 1–6, and the final yields of the products were in the 43–98% range. All compounds were fully characterized using standard methods, ¹H NMR, ¹³C NMR spectroscopy, and HRMS spectrometry. All NMR spectra are presented in the Supplementary Materials.

2.3. Biological Evaluation

2.3.1. 5-HT_1A Receptor Activity

After purification via column chromatography, all newly synthesized compounds were subjected to in vitro evaluation of their functional activity for the 5-HT_1A receptor, as well as 5-HT_2A receptor affinity studies. Since in our previous study similar coumarin derivatives showed high affinities to 5-HT_1A receptor and low to 5-HT_2A receptor we decided to employ in this study a functional assay to establish the potency and efficacy of 5-HT_1A binding of our series of compound. The major advantage of this approach over determining only receptor affinity is the ability to predict intracellular consequences of receptor binding, leading to either receptor activation, blockage, or alteration of constitutive activity. Moreover, measures of affinity may not correspond to drug potency, owing to the possible existence of a receptor reserve [27]. Also, for ligands displaying functional bias, measuring one distinctive activation pattern allows for the prediction of the therapeutic usefulness of the drug in question [28]. Thus, functional characteristics are of major importance for any drug discovery program which strives for in vivo evaluation of compound activity. For the 5-HT_2A receptor we expected, on the hand, low affinities and decided to perform standard receptor affinity studies.

As shown in

Table 3. Antagonistic activity of compounds for the 5-HT_1A receptor.

Compound	pIC50 ± SEM	EC50 (nM ± SEM)
1a	7.5 ± 0.21	29.4 ± 7.3
1b	6.0 ± 0.2	980 ± 207
1c	5.8 ± 0.22	1698 ± 358
1d	not tested	not tested
1e	4.6 ± 0.3	24,320 ± 3730
1f	4.7 ± 0.14	20,920 ± 2072
1g	not tested	not tested
1h	3.7 ± 0.18	198,100 ± 51,350
1i	4.8 ± 0.14	13,740 ± 1733
1j	4.1 ± 0.38	74,720 ± 23,990
2a	5.7 ± 0.18	1881 ± 427
2b	no activity	no activity
2c	4.7 ± 0.11	19,130 ± 2363
2d	no activity	no activity
2f	no activity	no activity
2h	no activity	no activity
2i	3.4 ± 1.4	365,800 ± 46,480
2j	no activity	no activity
3a	7.4 ± 0.17	39.4 ± 3.63
3b	4.7 ± 0.1	19,200 ± 1177
3c	5.8 ± 0.2	1549 ± 190
3d	5.0 ± 0.17	9434 ± 1037
3e	6.15 ± 0.11	702 ± 112
3f	6.16 ± 0.14	689 ± 138
3g	5.89 ± 0.12	1284 ± 254
3h	5.9 ± 0.16	1245 ± 112
3i	4.8 ± 0.11	15,400 ± 1290
3j	not tested	not tested
4a	7.0 ± 0.11	91.6 ± 13.3
4b	5.3 ± 0.13	5003 ± 218
4c	5.3 ± 0.07	5007 ± 117
4d	4.1 ± 0.04	74,730 ± 4576
4e	6.3 ± 0.18	527 ± 191
4f	5.08 ± 0.10	8317 ± 1497
4g	4.9 ± 0.11	11,070 ± 1395
4h	4.4 ± 0.11	26,840 ± 5904
4i	5.5 ± 0.15	3098 ± 148
4j	6.3 ± 0.06	538 ± 105
5a	7.5 ± 0.11	30.5 ± 2.56
5b	7.1 ± 0.13	82 ± 13.4
5c	4.6 ± 0.14	22,860 ± 2899
5d	4.9 ± 0.05	12,940 ± 802
5e	4.5 ± 0.12	33,890 ± 2563
5f	4.5 ± 0.11	27,440 ± 1986
5g	4.6 ± 0.13	21,520 ± 2347
5h	4.5 ± 0.14	32,890 ± 2417
5i	4.2 ± 0.41	55,920 ± 4987
5j	4.8 ± 0.22	13,860 ± 2059
6a	5.5 ± 0.1	2735 ± 626
6b	4.3 ± 0.07	45,330 ± 8178
6c	4.3 ± 0.09	47,150 ± 9511
6d	4.2 ± 0.11	59,890 ± 12,467
6e	4.0 ± 0.11	98,950 ± 15,583
6f	4.3 ± 0.07	46,850 ± 8125
6g	not tested	not tested
6h	4.3 ± 0.09	47,790 ± 9556
6i	3.7 ± 0.24	204,000 ± 56,920
6j	5.1 ± 0.1	7804 ± 1876
WAY-100635	8.4 ± 0.12	4.3 ± 0.86

, arylpiperazinyl derivatives of coumarin displayed varied selectivity for 5-HT_1A receptor with respect to WAY-100635, a reference compound which is a piperazine drug that acts as a selective 5-HT_1A receptor antagonist. The highest activity was found for compounds 1a, 3a, 4a, 5a, and 5b with the following values: 1a (EC₅₀ = 29.4 ± 7.3 nM) > 5a (EC₅₀ = 30.5 ± 2.56 nM) > 3a (EC₅₀ = 39.4 ± 3.63 nM) > 5b (EC₅₀ = 82 ± 13.4 nM) > 4a (EC₅₀ = 91.6 ± 13.3 nM). Compounds 2b, 2d, 2f, 2h, and 2j did not show any activity and four compounds 1d, 1g, 3j, and 6g were not tested due to a very poor solubility under experimental conditions. The remaining coumarin derivatives showed moderate to low activity ranging from EC₅₀ = 527 ± 191 nM for compound 4e, to EC₅₀ = 365,800 ± 46,480 nM for compound 2i.

The structure–activity studies revealed that the presence of a (2-methoxyphenyl) piperazine moiety and a five carbon linker (1a, 3a, 4a, 5a) was the most beneficial for 5-HT_1A antagonistic activity. This was a trend independent of the starting coumarin derivative, as a high antagonistic activity was obtained for 5-hydroxy-4,7-dimethylchromen-2-one derivative (1a), 6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one (3a), and 8-acetyl-7-hydroxy-4-methylchromen- 2-one (5a). Only one compound with a two carbon linker, namely 4a (6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one), showed a similarly high level of activity. Also, a high activity was found for one derivative bearing a (2-fluorophenyl) piperazine moiety, 8-acetyl-7-hydroxy-4-methylchromen-2-one (5b).

In the family of 5-hydroxy-4,7-dimethylchromen-2-one (A) derivatives, compounds with a five carbon linker were much more active than those with a two carbon linker. Comparing the systems containing the same piperazinyl part within this family, we can see that the five carbon linker derivatives have always a higher activity than the one with two CH₂ moieties, e.g., EC₅₀ = 29.4 ± 7.3 nM for 1a and EC₅₀ = 1881 ± 427 nM for 2a; EC₅₀ = 980 ± 207 nM for 1b and no activity for 2b; EC₅₀ = 1698 ± 358 nM for 1c and EC₅₀ = 19130 ± 2363 nM for 2c, etc. For 6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one (B) derivatives, which differ from the A family in the presence of an additional acetyl group at the position C-6 of the coumarin ring, derivatives with the five-carbon linker had higher activity than those with the two-carbon linker, when they contained 2-methoxyphenyl (see 3a and 4a), 3-methoxyphenyl (see 3c and 4c), 2,5-dimethylyphenyl (see 3d and 4d), 3-bromophenyl (see 3g and 4g), or 2,5-dimethylyphenyl moiety (see 3h and 4h). On the other hand, derivatives with the two-carbon linker showed higher activity than those with the five-carbon linkers, when they contained the 2-fluorophenyl (see 3b and 4b), 3-fluorophenyl (see 3e and 4e), 2-bromophenyl (see 3f and 4f), or 2,3-dichlorophenyl moiety (see 3i and 4i). Such a difference may stem from the fact that a longer alkyl linker may maximize the interactions of the ligand with the receptor’s residues of different transmembrane regions for all derivatives apart from selected B derivatives, which due to the presence of the additional acetyl moiety makes the ligand too large for bulkier arylpiperazines to find optimal interactions in the binding site. Molecular docking studies suggest that upon anchoring to D116 coumarin derivatives can extend both toward transmembrane regions 4 and 7 to find favorable interactions within the binding site. The two-carbon linker makes such an extension impossible, lowering in most cases the potency of the antagonist.

Finally, for 8-acetyl-7-hydroxy-4-methylchromen-2-one (C) derivatives, all compounds with the five-carbon linker (5a–5i) showed higher antagonistic activities than their two-carbon linker counterparts (6a–6i), with the exception of 8-acetyl-7-(2-[4-(2-cyanophenyl)piperazin- 1-yl]ethoxy)-4-methylchromen-2-one (6j), which showed a higher activity (EC₅₀ = 7804 ± 1876 nM) than its analogue, 8-acetyl-7-(5-[4-(2-cyanophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5j) (EC₅₀ = 13,860 ± 2059 nM). We can speculate that the higher antagonistic activities are a result of a similar structural feature as in the A family, due to a different position of the acetyl moiety in the C family, which lowers the volume of these derivatives with respect to the B family.

There is also a group of compounds with a moderately high 5-HT_1A antagonistic activity, which consists of 5-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1b), 6-acetyl-5-(5-(4-(3-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3e), 6-acetyl-5-(5-(4-(2-bromophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3f), 6-acetyl-5-(2-(4-(3-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4e) and 6-acetyl-5-(2-(4-(2-cyanophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4j) (EC₅₀ = 980 ± 207 nM, EC₅₀ = 702 ± 112 nM, EC₅₀ = 689 ± 138 nM, EC₅₀ = 527 ± 191 nM, and EC₅₀ = 538 ± 105 nM, respectively). There is no one particular shared structural feature of this group of compounds as it is composed of both 5-(CH₂) (1b, 3e, and 3f) and 2-(CH₂) (4e, 4j) linkers and various arylpiperazines (2 or 3-fluorophenyl, 2-bromophenyl, or 2-cyanophenyl).

Two compounds, namely 5-(2-(4-(3-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl -2H-chromen-2-one (2e) and 5-(4-(4-(3-bromophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl- 2H-chromen-2-one (2g) acted as weak partial agonists at the 5-HT1A receptor. (

Table 4. G-protein enhancing effect of compounds 2e and 2f at the 5-HT_1A receptor.

Compound	pEC50	EC50 (nM ± SEM)	Emax (% ± SEM)
2e	6.7 ± 0.25	178.7 ± 39.3	118 ± 1.48
2g	5.2 ± 0.73	5083 ± 2636	113 ± 6.5

). Interestingly, both of these derivatives contain a two-carbon linker between the arylpiperazinyl and the coumarin core and the phenyl ring on the piperazine with a halogen atom in position C-3. In the case of compound 2e (Emax = 118 ± 1.48) it is a fluorine atom, while in the case of 2g (Emax = 118 ± 6.5) it is a bromine atom.

2.3.2. 5-HT_2A Receptor Affinity

As, it was shown in our previous studies, coumarin derivatives containing the three-carbon or four-carbon linker between the coumarin and piperazine moieties showed moderate affinities for the 5-HT_2A receptor [19,20,21]. As shown in

Table 5. 5-HT_2A receptor binding of investigated compounds.

Compound	pKi (M ± SEM)	Ki (nM ± SEM)
1a	6.191 ± 0.12	776 ± 187
1b	5.90 ± 0.2	1263 ± 479
1c	6.21 ± 0.13	610 ± 139
1d	not tested	not tested
1e	5.63 ± 0.16	2346 ± 673
1f	5.81 ± 0.09	1548 ± 323
1g	not tested	not tested
1h	5.74 ± 0.14	1818 ± 636
1i	5.84 ± 0.11	1452 ± 653
1j	7.10 ± 0.12	79 ± 18
2a	5.77 ± 0.15	1713 ± 582
2b	5.67 ± 0.11	2129 ± 662
2c	6.33 ± 0.10	458 ± 109
2d 2e	5.13 ± 0.12 not tested	7321 ± 2079 not tested
2f 2g	5.02 ± 0.12 5.45 ± 0.11	9600 ± 2841 3538 ± 1135
2h	5.53 ± 0.13	2920 ± 992
2i	5.37 ± 0.17	4306 ± 1550
2j	5.25 ± 0.14	5619 ± 1854
3a	6.19 ± 0.12	641 ± 128
3b	5.93 ± 0.11	1223± 166
3c	6.31 ± 0.14	492 ± 99.7
3d	5.71 ± 0.14	1938 ± 388
3e	5.85 ± 0.09	1421 ± 426.3
3f	6.45 ± 0.09	354 ± 110
3g	6.18 ± 0.11	664 ± 154
3h	5.75 ± 0.13	1796 ± 521
3i	6.31 ± 0.09	486 ± 102
3j	not tested	not tested
4a	5.28 ± 0.13	5214 ± 1246
4b	5.15 ± 0.12	7053 ± 1650
4c	6.29 ± 0.15	513 ± 137
4d	4.93 ± 0.14	11,870 ± 3086
4e	5.41 ± 0.11	3859 ± 810
4f	4.97 ± 0.16	10,610 ± 3045
4g	5.40 ± 0.07	3986 ± 745
4h	5.72 ± 0.08	1924 ± 382
4i	5.78 ± 0.13	1642 ± 344
4j	5.61 ± 0.04	2429 ± 374
5a	6.46 ± 0.11	343 ± 86
5b	6.91 ± 0.17	122 ± 43
5c	6.84 ± 0.13	144 ± 38
5d	5.82 ± 0.08	1497 ± 279
5e	5.67 ± 0.09	2114 ± 420
5f	6.96 ± 0.10	108 ± 24
5g	7.09 ± 0.13	81 ± 19
5h	6.54 ± 0.09	291 ± 57
5i	7.20 ± 0.07	51 ± 8.3
5j	5.59 ± 0.13	2551 ± 765
6a	6.12 ± 0.11	752 ± 171
6b	5.82 ± 0.11	1509 ± 344
6c	6.03 ± 0.09	920 ± 160
6d	5.69 ± 0.10	2046 ± 405
6e	5.78 ± 0.08	1671 ± 274
6f	5.82 ± 0.08	1503 ± 268
6g	5.83 ± 0.12	1494 ± 466
6h	6.32 ± 0.11	479 ± 96
6i	6.12 ± 0.11	755 ± 178
6j	6.30 ± 0.11	496 ± 121
ketanserin	8.44 ± 0.07	3.6 ± 0.5

, arylpiperazinyl derivatives of coumarin containing the two-carbon or five-carbon carbon linkers displayed varied 5-HT_2A receptor binding, but none of them showed affinities comparable to the reference compound, katanserin (K_i = 3.6 ± 0.5 nM). The highest binding was found for compounds 5i, 1j, and 5g (K_i = 51 ± 8.3 nM, 79 ± 18 nM and 81 ± 19 nM, respectively). Compounds 5b, 5c, and 5f showed moderate binding ranging from K_i = 108 ± 24 nM for compound 5f, K_i = 122 ± 43 nM for compound 5b, to K_i = 144 ± 38 nM for compound 5c. The remaining compounds showed weak 5-HT_2A receptor binding, ranging between K_i = 291 ± 57 nM for compound 5h and K_i = 11,870 ± 3086 nM for compound 4d.

Overall, for the 5-HT_2A receptor, the group of 8-acetyl-7-hydroxy-4-methylchromen-2-one (C) derivatives with the five-carbon linker (compounds 5a–5j) turned out to be the most promising ones. In this group we found compounds with the highest (5i, 5g) and moderate affinity (5b, 5c, 5f). All derivatives with the five-carbon linker in the remaining families (A nad B) showed rather weak affinities, except 5-(5-(4-(2-cyanophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1j), from the 5-hydroxy-4,7-dimethylchromen-2-one (A) group of compounds. All derivatives containing the two-carbon linker also showed rather weak affinities for the 5-HT_2A receptor. These results indicate that only compounds bearing the (2-cyanophenyl)piperazin-1-yl (1j), (2,2-dichloro)piperazin-1-yl (5i) or (3-bromophenyl)piperazin-1-yl (5g) moieties were able to bind to the 5-HT_2A with high affinities. The introduction of the 2-cyano group to the phenyl ring of piperazine increased the 5-HT_2A receptor affinity in the case of 5-hydroxy- 4,7-dimethylchromen-2-one (A). On the other hand, introduction of the same cyano moiety to the phenyl ring of piperazine in 8-acetyl-7-hydroxy-4-methylchromen-2-one (C), drastically decreased binding from K_i = 79 ± 18 for 1j to K_i = 2551 ± 765 for 5j. The introduction of the 2,3-dichloro or 3-bromo moiety to phenyl ring of piperazine increased the affinity when the coumarin moiety was 8-acetyl-7-hydroxy-4-methyl chromen-2-one (C). In the case of 5-hydroxy-4,7-dimethyl chromen-2-one (A) and 6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one (B) derivatives, introduction of the 2,3-dichloro or 3-bromo moieties resulted in derivatives with weak 5-HT_2A receptor affinities as seen for 1i, 2i, 3i, 4i, 6i (K_i = 1452 ± 653, K_i = 4306 ± 1550, K_i = 486 ± 102, K_i = 1642 ± 344 and K_i = 755 ± 178, respectively)) and 2g, 3g, 4g, 6g (K_i = 3538 ± 1135, K_i = 664 ± 154, K_i = 3986 ± 745 and K_i = 1494 ± 466, respectively). Changing the C-3 position of the bromo substituent on the phenyl ring of piperazine to the C-2 position slightly decreased binding affinity from K_i = 81 ± 19 for 8-acetyl-7-(5-[4-(3-bromohenyl)piperazin-1-yl]penthoxy)- -4-methylchromen-2-one (5g) to K_i = 108 ± 24 for 8-acetyl-7-(5-[4-(2-bromophenyl)piperazin-1-yl]- penthoxy)-4-methyl chromen-2-one (5f). Converting the bromine atom at the C-3 position to a fluorine atom drastically decreased binding affinity from K_i = 81 ± 19 for 5g to K_i = 2114 ± 420 for 5e. Similarly, the replacement of the cyano group at the C-2 position with a fluoro or bromo moiety at the C-2 position resulted in a decrease in affinity for the 5-HT_2A receptor, from K_i = 79 ± 18 for 1j to K_i = 1263 ± 479 for 1b and K_i = 1548 ± 323 for 1f.

The moderate agreement between the experimental and theoretical K_i values for 5HT_2A receptor warrant a short comment. While the predicted K_i values for the newly synthesized set of coumarins derivatives are usually in the low nanomolar range, the experimental values are usually closer to micromolar values. The most likely explanation of these discrepancies is the combination of the imperfection of our computational model of the 5HT_2A receptor, particularly in the binding site part and the limited accuracy of the computational methods. The second problem is very well-known in the scientific community, as it has been shown that while Autodock and other similar programs can identify the correct binding poses, they often have problem is predicting correct bonding affinities [29]. As for the accuracy of homology models of GPCRs, they certainly can be improved by resorting to more sophisticated methods, such as e.g., using multiple templates or going beyond the homology modelling, and we are planning to make use of these new methods in the future [30,31,32]. Nevertheless the most 2D schematic representations of the predicted binding sites for the selects, most interesting coumarins derivatives are presented in the Supplementary Materials. Taking compound 1j as the example we can suggest, that this compound is able to perfectly fit into the binding site of the 5HT_2A receptor, keeping the salt bridge to D155, while retaining the coumarin part in the hydrophobic region of the binding site and the piperazine part in the hydrophilic one. This is not true for this compound binding to the 5HT_1A receptor, as the salt bridge to D116 forced 1j to move the coumarin part into a more hydrophilic region, lowering the affinity to the receptor. Additionally, 1j in the binding site of the 5HT_2A receptor is stabilized by two hydrogen bonds and a π-π stacking interactions with F340.

3. Materials and Methods

All starting materials were purchased from Aldrich or Merck and used without further purification. Microwave oven Plazmatronika 1000 was used (http://www.plazmatronika.com.pl (accessed on 27 December 2020)). Melting points were determined with ElectroThermal 9001 Digital Melting Point apparatus and are uncorrected. High resolution mass spectra were recorded on Quattro LCT (TOF). ¹H NMR, ¹³C NMR spectra in solution were recorded at 25 C with a Varian NMRS-300 spectrometer, and standard Varian software was employed. The calculated shielding constants were used as an aid in an assignment of resonances of ¹³C atoms. Chemical shifts d [ppm] were referenced to TMS. TLC was carried out using Kieselgel 60 F254 sheets and spots were visualized by UV e 254 and 365 nm.

3.1. Chemistry

Compounds 1–6 and 1a–1j, 2a–2j, 3a–3j, 4a–4j, 5a–5j, 6a–6j were prepared in accordance with the previously reported procedures [19,33]. Atom numbering, ¹H NMR and ¹³C NMR spectra of all synthesized compounds are available in the ESI.

5-(5-bromopentyloxy)-4,7-dimethyl-2H-chromen-2-one (1). Yield 44%; white solid; m.p. 99–101 °C; Rf = 0.86; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.74 (1H, s, H-8), 6.51 (1H, s, H-6), 6.05 (1H, s, H-3), 4.04 (2H, t, J = 8 Hz, H-1′), 3.46 (2H, t, J = 8 Hz, H-5′), 2.58 (3H, s, H-10), 2.39 (3H, s, H-9), 2.01–1.85 (4H, m, H-2′, H-4′), 1.73–1.60 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.4 (C-5), 155.5 (C-4), 154.2 (C-8a), 143.2 (C-7), 113.7 (C-4a), 110.4 (C-6), 108.4 (C-3), 108.0 (C-8), 68.8 (C-1′), 33.6 (C-5′), 33.2 (C-4′), 32.5 (C-2′), 28.4 (C-3′), 25.2 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₁₆H₁₉O₃Na Br (361.0415) found 361.0401.

5-(5-(4-(2-methoxyphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1a). Yield 90%; white solid; m.p. 66–68 °C; Rf = 0.16; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.03–6.85 (4H, m, H-3”, H-4”, H-5”, H-6”), 6.73 (1H, s, H-8), 6.51 (1H, s, H-6), 6.04 (1H, s, H-3), 4.03 (2H, t, J = 10 Hz, H-1′), 3.86 (3H, s, H-7”), 3.11 (4H, br. s., H-3p, H-5p), 2.67 (4H, br. s., H-2p, H-6p), 2.58 (3H, s, H-10), 2.46 (2H, t, J = 10 Hz, H-5′), 2.38 (3H, s, H-9), 1.92–1.88 (2H, m, H-2′), 1.64–1.55 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-1”), 157.4 (C-2), 155.5 (C-4), 154.3 (C-5), 152.4 (C-8a), 143.2 (C-7, C-2”), 123.4 (C-6”), 121.2 (H-4”), 118.5 (C-5”), 113.6 (C-4a), 111.4 (C-6), 110.3 (C-3), 108.4 (C-3”), 118.1 (C-8), 68.9 (C-1′), 58.5 (C-3p, C-5p), 55.6 (C-5′), 53.5 (C-7”), 50.2 (C-2p, C-6p), 29.2 (C-2′), 24.7 (C-4′), 24.4 (C-10, C-3′), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₇H₃₅O₄N₂ (451.2597) found 451.2583.

5-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1b). Yield 91%; white solid; m.p. 106–108 °C; Rf = 0.20; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.09–6.89 (4H, m, H-3”, H-4”, H-5”, H-6”), 6.75 (1H, s, H-8), 6.51 (1H, s, H-6), 6.04 (1H, s, H-3), 4.03 (2H, t, J = 8 Hz, H-1′), 3.12 (4H, t, J = 6 Hz, H-3p, H-5p), 2.64 (4H, t, J = 6 Hz, H-2p, H-6p), 2.58 (3H, s, H-10), 2.45 (2H, t, J = 10 Hz, H-5′), 2.38 (3H, s, H-9), 1.94–1.87 (2H, m, H-2′), 1.66–1.52 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.5 (C-2”), 157.4 (C-4), 155.5 (C-5), 154.2 (C-8a), 143.2 (C-7), 140.1 (C-1”), 124.7 (C-5”), 122.8 (C-4”), 119.2 (C-3”), 116.4 (C-6”), 116.2 (C-4a), 113.6 (C-6), 110.3 (C-3), 108.0 (C-8), 68.9 (C-1′), 58.5 (C-3p, C-5p), 53.4 (C-5′), 50.4 (C-2p, C-6p), 29.2 (C-2′), 26.4 (C-4′), 24.7 (C-3′), 24.4 (C-10), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₆H₃₂O₃N₂F (439.2397) found 439.2403.

5-(5-(4-(3-methoxyphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1c). Yield 84%; white solid; m.p. 103–105 °C; Rf = 0.31; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.17 (1H, t, J = 12 Hz, H-5”), 6.74 (1H, s, H-8), 6.51–6.43 (4H, m, H-6, H-4”, H-2”, H-6”), 6.04 (1H, s, H-3), 4.03 (2H, t, J = 8 Hz, H-1′), 3.79 (3H, s, H-7”), 3.21 (4H, t, J = 8 Hz, H-3p, H-5p), 2.62–2.58 (7H, m, H-10, H-2p, H-6p), 2.43 (2H, t, J = 8 Hz, H-5′), 2.38 (3H, s, H-9), 1.94–1.85 (2H, m, H-2′), 1.68–1.51 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-3”), 160.8 (C-2), 157.4 (C-4), 155.5 (C-5), 154.2 (C-8a, C-1”), 143.2 (C-7), 130.1 (C-5”), 113.6 (C-4a), 110.3 (C-6), 109.2 (C-3), 108.4 (C-4”), 108.0 (C-8), 105.0 (C-6”), 103.0 (C-2”), 68.9 (C-1′), 58.4 (C-3p, C-5p), 55.4 (C-5′), 53.1 (C-7”), 48.7 (C-2p, C-6p), 29.2 (C-2′), 24.8 (C-4′, C-3′), 24.4 (C-10), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₇H₃₅O₄N₂ (451.2597) found 451.2585.

5-(5-(4-(2,5-dimethylphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1d). Yield 82%; white solid; m.p. 132–134 °C; Rf = 0.29; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.05 (1H, d, J = 12 Hz, H-3”), 6.83–6.78 (2H, m, H-6, H-4”), 6.74 (1H, s, H-6”), 6.52 (1H, s, H-8), 6.05 (1H, s, H-3), 4.04 (2H, t, J = 8 Hz, H-1′), 2.95 (4H, t, J = 6 Hz, H-3p, H-5p), 2.62–2.59 (7H, m, H-2p, H-6p, H-10), 2.46 (2H, t, J = 10 Hz, H-5′), 2.39 (3H, s, H-9), 2.30 (3H, s, H-7”), 2.25 (3H, s, H-8”), 1.93–1.88 (2H, m, H-2′), 1.60–1.55 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.4 (C-4), 155.5 (C-5), 154.2 (C-8a, C-1”), 143.2 (C-7), 136.6 (C-5”), 131.1 (C-2”), 129.4 (H-3”), 124.7 (C-4′), 120.3 (C-4a), 113.6 (C-6), 110.3 (C-3), 108.4 (C-6”), 108.0 (C-8), 68.8 (C-1′, C-5′), 58.3 (C-3p, C-5p), 51.8 (C-2p, C-6p), 29.1 (C-2′), 24.8 (C-4′), 24.3 (C-3′), 22.2 (C-10), 21.3 (C-9), 17.6 (C-7”, C-8”); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₇O₃N₂ (449.2804) found 449.2790.

5-(5-(4-(3-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1e). Yield 77%; white solid; m.p. 101–102 °C; Rf = 0.28; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.22–7.14 (1H, m, H-5”), 6.73 (1H, s, H-2”), 6.68–6.51 (4H, m, H-6, H-8, H-4”, H-6”), 6.04 (1H, s, H-3), 4.03 (2H, t, J = 8 Hz, H-1′), 3.21 (4H, t, J = 6 Hz, H-3p, H-5p), 2.61–2.57 (7H, m, H-10, H-2p, H-6p), 2.43 (2H, t, J = 10 Hz, H-5′), 2.38 (3H, s, H-9), 1.92–1.87 (2H, m, H-2′), 1.60–1.54 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 165.7 (C-3”), 161.2 (C-2), 157.5 (C-4), 155.5 (C-5), 154.3 (C-1”), 153.0 (C-8a), 143.2 (C-7), 130.4 (C-5”), 113.6 (C-4a), 111.2 (C-6), 110.3 (C-3), 108.4 (C-6”), 106.2 (C-4”),105.9 (C-8), 102.6 (C-2”), 69.0 (C-1′), 58.6 (C-3p, C-5p), 53.3 (C-5′), 48.8 (C-2p, C-6p), 29.3 (C-2′), 26.8 (C-4′), 24.8 (C-10), 24.5 (C-3′), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₆H₃₂O₃N₂F (439.2386) found 439.2391.

5-(5-(4-(2-bromophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1f). Yield 48%; white solid; m.p. 143–144 °C; Rf = 0.34; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.56 (1H, dd, J = 12 Hz, H-3”), 7.30–7.24 (1H, m, H-5”), 7.05 (1H, dd, J = 12 Hz, H-4”), 6.91 (1H, t, J = 12 Hz, H-6”), 6.74 (1H, s, H-8), 6.52 (1H, s, H-6), 6.04 (1H, s, H-3), 4.04 (2H, t, J = 8 Hz, H-1′), 3.08 (4H, br. s., H-3p, H-5p), 2.66 (4H, br. s., H-2p, H-6p), 2.59 (3H, s, H-10), 2.47 (2H, t, J = 10 Hz, H-5′), 2.39 (3H, s, H-9), 1.93–1.88 (2H, m, H-2′), 1.60–1.55 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.5 (C-4), 155.5 (C-5), 154.3 (C-8a, C-1”), 143.2 (C-7), 134.0 (C-3”), 128.5 (C-4”), 124.6 (H-5”), 121.2 (C-6”, C-2”), 120.0 (C-4a), 113.6 (C-6), 110.3 (C-3), 108.0 (C-8), 69.0 (C-1′), 58.6 (C-3p, C-5p), 53.6 (C-5′), 51.8 (C-2p, C-6p), 29.3 (C-2′), 24.8 (C-4′), 24.5 (C-3′, C-10), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₆H₃₂O₃N₂Br (499.1596) found 499.1594.

5-(5-(4-(3-bromophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1g). Yield 66%; white solid; m.p. 107–109 °C; Rf = 0.26; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.13–7.07 (1H, m, H-4”), 7.03 (1H, t, J = 4 Hz, H-5”), 6.96–6.93 (1H, m, H-6”), 6.84–6.81 (1H, m, H-2”), 6.74 (1H, s, H-8), 6.51 (1H, s, H-6), 6.04 (1H, s, H-3), 4.04 (2H, t, J = 10 Hz, H-1′), 3.20 (4H, t, J = 6 Hz, H-3p, H-5p), 2.61–2.58 (7H, m, H-10, H-2p, H-6p), 2.43 (2H, t, J = 8 Hz, H-5′), 2.38 (3H, s, H-9), 1.92–1.87 (2H, m, H-2′), 1.59–1.54 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.5 (C-4), 155.5 (C-5), 154.3 (C-1”), 152.6 (C-8a), 143.2 (C-7), 130.5 (C-5”), 123.4 (C-3”), 122.4 (C-4”), 118.8 (C-4a), 114.5 (C-6), 113.6 (C-2”), 110.3 (C-6”), 108.4 (C-3), 108.0 (C-8), 69.0 (C-1′), 58.6 (C-3p, C-5p), 53.3 (C-5′), 48.8 (C-2p, C-6p), 29.3 (C-2′), 26.7 (C-4′), 24.8 (C-10), 24.5 (C-3′), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₆H₃₂O₃N₂Br (499.1596) found 439.1612.

5-(5-(4-(3,5-dimethylphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1h). Yield 65%; white solid; m.p. 104–106 °C; Rf = 0.34; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.74 (1H, s, H-8), 6.56 (2H, s, H-2”, H-6”), 6.52 (2H, s, H-4”, H-6), 6.04 (1H, s, H-3), 4.03 (2H, t, J = 8 Hz, H-1′), 3.19 (4H, t, J = 8 Hz, H-3p, H-5p), 2.62–2.58 (7H, m, H-2p, H-6p, H-10), 2.43 (2H, t, J = 10 Hz, H-5′), 2.38 (3H, s, H-9), 2.27 (6H, s, H-7”, H-8”), 1.92–1.87 (2H, m, H-2′), 1.63–1.54 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.5 (C-4), 155.5 (C-5), 154.3 (C-8a), 151.6 (C-1”), 143.2 (C-7), 136.8 (C-3”, C-5”), 121.9 (C-4”), 114.2 (C-4a), 113.6 (C-2”, C-6”), 110.3 (C-6), 108.4 (C-3), 108.0 (C-8), 69.0 (C-1′), 58.7 (C-3p, C-5p), 53.6 (C-5′), 49.4 (C-2p, C-6p), 29.3 (C-2′), 26.8 (C-4′), 24.8 (C-3′), 24.5 (C-10), 22.2 (C-9), 21.9 (C-7”, C-8”); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₇O₃N₂ (449.2804) found 449.2809.

5-(5-(4-(2,3-dichlorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1i). Yield 62%; white solid; m.p. 162–163 °C; Rf = 0.38; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.16–7.14 (2H, m, H-4”, H-5”), 6.97–6.94 (1H, m, H-6”), 6.74 (1H, s, H-8), 6.52 (1H, s, H-6), 6.05 (1H, s, H-3), 4.04 (2H, t, J = 8 Hz, H-1′), 3.09 (4H, t, br. s., H-3p, H-5p), 2.66 (4H, br. s., H-2p, H-6p), 2.59 (3H, s, H-10), 2.47 (2H, t, J = 8 Hz, H-5′), 2.39 (3H, s, H-9), 1.93–1.88 (2H, m, H-2′), 1.60–1.57 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.5 (C-4), 155.5 (C-5), 154.3 (C-8a, C-1”), 143.2 (C-7), 134.3 (C-3”), 127.2 (C-5”), 124.9 (C-2”), 118.8 (C-4”), 113.7 (C-6”), 110.3 (C-4a), 108.4 (C-3, C-6), 108.0 (C-8), 69.0 (C-1′), 58.6 (C-3p, C-5p), 53.5 (C-5′), 51.3 (C-2p, C-6p), 29.3 (C-2′), 26.6 (C-4′), 24.8 (C-3′), 24.5 (C-10), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₆H₃₁O₃N₂Cl₂ (489.1712) found 489.1695.

5-(5-(4-(2-cyanophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (1j). Yield 84%; cream solid; m.p. 148–149 °C; Rf = 0.32; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.56 (1H, d, J = 8 Hz, H-3”), 7.49 (1H, t, J = 10 Hz, H-5”), 7.02–7.00 (2H, m, H-4”, H-6”), 6.47 (1H, s, H-8), 6.52 (1H, s, H-6), 6.04 (1H, s, H-3), 4.02 (2H, t, J = 8 Hz, H-1′), 3.25 (4H, t, J = 6 Hz, H-3p, H-5p), 2.68 (4H, br. s., H-2p, H-6p), 2.58 (3H, s, H-10), 2.47 (2H, t, J = 10 Hz, H-5′), 2.39 (3H, s, H-9), 1.93–1.88 (2H, m, H-2′), 1.64–1.55 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.2 (C-2), 157.5 (C-4), 155.8 (C-5), 155.5 (C-8a), 154.3 (C-1”), 143.2 (C-7), 134.6 (C-5”), 134.0 (C-3”), 122.0 (C-7”), 118.9 (C-4”), 118.6 (C-6”), 113.6 (C-4a), 110.3 (C-6), 108.4 (C-7), 108.0 (C-3), 106.3 (C-8, C-2”), 69.0 (C-1′), 58.5 (C-3p, C-5p), 53.4 (C-5′), 51.6 (C-2p, C-6p), 29.3 (C-3′), 26.6 (C-4′), 24.5 (C-10), 22.2 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₇H₃₂O₃N₃ (446.2444) found 446.2455.

5-(2-bromoethoxy)-4,7-dimethyl-2H-chromen-2-one (2). Yield 25%; white solid; m.p. 121–123 °C; Rf = 0.86; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.81 (1H, s, H-8), 6.50 (1H, s, H-6), 6.10 (1H, s, H-3), 4.40 (2H, t, J = 8 Hz, H-1′), 3.74 (2H, t, J = 8 Hz, H-2′), 2.66 (3H, s, H-10), 2.41 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.0 (C-2), 156.4 (C-4), 155.6 (C-5), 154.2 (C-8a), 143.2 (C-7), 114.1 (C-4a), 111.2 (C-6, C-3), 107.9 (C-8), 68.8 (C-1′), 29.0 (C-2′), 24.9 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₁₃H₁₃O₃Na Br (318.9946) found 318.9961.

5-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2a). Yield 70%; cream solid; m.p. 146–148 °C; Rf = 0.72; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.04–6.86 (4H, m, H-3”, H-4”, H-5”, H-6”), 6.76 (1H, s, H-8), 6.56 (1H, s, H-6), 6.05 (1H, s, H-3), 4.22 (2H, t, J = 6 Hz, H-1′), 3.87 (3H, s, H-7”), 3.13 (4H, br. s., H-3p, H-5p), 2.95 (2H, t, J = 10 Hz, H-2′), 2.80 (4H, br. s., H-2p, H-6p), 2.62 (3H, s, H-10), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-1”), 157.2 (C-2), 155.5 (C-4), 154.4 (C-5), 152.5 (C-8a), 143.2 (C-2”), 141,6 (C-7), 123.3 (C-6”), 121.2 (H-5”), 118.4 (C-4”, C-3”), 113.7 (C-4a), 111.4 (C-6), 110.6 (C-3), 108.3 (C-8), 66.8 (C-1′), 57.2 (C-3p, C-5p), 55.6 (C-2′), 53.9 (C-7”), 50.8 (C-2p, C-6p), 24.8 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₄H₂₈O₄N₂Na (431.1947) found 431.1954.

5-(2-(4-(2-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2b). Yield 98%; white solid; m.p. 121–123 °C; Rf = 0.82; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.09–7.01 (2H, m, H-3”, H-5”), 6.99–6.90 (2H, m, H-4”, H-6”), 6.74 (1H, s, H-8), 6.55 (1H, s, H-6), 6.04 (1H, s, H-3), 4.19 (2H, t, J = 8 Hz, H-1′), 3.22–3.20 (4H, m, H-3p, H-5p), 2.93 (2H, t, J = 8 Hz, H-2′), 2.77 (4H, t, J = 8 Hz, H-2p, H-6p), 2.61 (3H, s, H-10), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.5 (C-2”), 157.2 (C-4), 155.4 (C-5), 154.2 (C-8a), 143.2 (C-7), 140.2 (C-1”), 124.7 (C-5”), 124.6 (C-4a), 122.8 (C-4”), 119.1 (C-3′), 116.4 (C-6”), 113.6 (C-6), 110.5 (C-3), 108.5 (C-8), 66.8 (C-1′), 57.2 (C-2′), 53.8 (C-3p, C-5p), 50.7 (C-2p, C-6p), 24.7 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₃H₂₅O₃N₂FNa (419.1747) found 419.1729.

5-(2-(4-(3-methoxyphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2c). Yield 89%; cream solid; m.p. 124–125 °C; Rf = 0.70; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.17 (1H, t, J = 16 Hz, H-5”), 6.75 (1H, s, H-8), 6.55–6.53 (2H, m, H-6, H-6”), 6.47–6.41 (2H, m, H-2”, H-4”), 6.04 (1H, s, H-3), 4.19 (2H, t, J = 8 Hz, H-1′), 3.97 (3H, s, H-7”), 3.53 (4H, t, J = 6 Hz, H-3p, H-5p), 2.91 (2H, t, J = 8 Hz, H-2′), 2.73 (4H, t, J = 6 Hz, H-2p, H-6p), 2.60 (3H, s, H-10), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-3”), 160.8 (C-2), 157.1 (C-4), 155.5 (C-5), 154.3 (C-8a), 152.7 (C-1”), 143.2 (C-7), 129.9 (C-5”), 113.7 (C-4a), 110.6 (C-6), 109.1 (C-3), 108.6 (C-4”), 108.3 (C-8), 104.7 (C-6”), 102.8 (C-2”), 66.8 (C-1′), 57.2 (C-2′), 55.4 (C-3p, C-5p), 53.7 (C-7”), 49.3 (C-2p, C-6p), 24.8 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₄H₂₈O₄N₂Na (431.1947) found 431.1929.

5-(2-(4-(2,5-dimethylphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2d). Yield 74%; cream solid; m.p. 119–120 °C; Rf = 0.90; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.05 (1H, d, J = 12 Hz, H-3”), 6.83–6.79 (2H, m, H-6”, H-2”), 6.74 (1H, s, H-8), 6.56 (1H, s, H-6), 6.04 (1H, s, H-3), 4.20 (2H, t, J = 8 Hz, H-1′), 2.96–2.91 (6H, m, H-3p, H-5p, H-2′), 2.73 (4H, br. s, H-2p, H-6p), 2.62 (3H, s, H-9), 2.39 (3H, s, H-10), 2.30 (3H, s, H-7′), 2.26 (3H, s, H-8”); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.2 (C-4), 155.5 (C-5), 154.4 (C-8a), 151.3 (C-1”), 143.2 (C-7), 136.3 (C-5”), 131.1 (C-2”), 129.4 (H-3”), 124.0 (C-4”), 119.9 (C-4a), 113.7 (C-6”), 110.5 (C-6), 108.6 (C-3), 108.3 (C-8), 66.8 (C-1′), 57.2 (C-2′), 54.3 (C-3p, C-5p), 51.9 (C-2p, C-6p), 24.7 (C-10), 22.2 (C-9), 21.4 (C-8”), 17.6 (C-7”); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₃₀O₃N₂Na (429.2154) found 429.2164.

5-(2-(4-(3-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2e). Yield 90%; brown solid; m.p. 120–122 °C; Rf = 0.80; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.19 (1H, q, H-5”), 6.74 (1H, s, H-2”), 6.69–6.60 (2H, m, H-8, H-6”), 6.55–6.49 (2H, m, H-6, H-4”), 6.03 (1H, s, H-3), 4.18 (2H, t, J = 6 Hz, H-1′), 3.22 (4H, t, J = 6 Hz, H-3p, H-5p), 2.91 (2H, t, J = 8 Hz, H-2′), 2.72 (4H, t, J = 6 Hz, H-2p, H-6p), 2.60 (3H, s, H-10), 2.39 (3H, s, H-10); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 165.6 (C-3”), 162.4 (C-2), 161.1 (C-4), 155.5 (C-5), 154.3 (C-1”), 153.0 (C-8a), 143.2 (C-7), 130.4 (C-5”), 113.7 (C-4a), 111.3 (C-6), 110.5 (C-3), 108.5 (C-4”), 108.3 (C-6”),106.3 (C-8), 103.0 (C-2”), 66.7 (C-1′), 57.1 (C-2′), 53.5 (C-3p, C-5p), 48.8 (C-2p, C-6p), 24.7 (C-10), 22.1 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₃H₂₅O₃N₂FNa (419.1747) found 419.1761.

5-(2-(4-(2-bromophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2f). Yield 75%; yellow solid; m.p. 129–130 °C; Rf = 0.93; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.56 (1H, dd, J₁ = 4 Hz, J₂ = 12 Hz, H-3”), 7.30–7.25 (1H, m, H-5”), 7.10–7.04 (1H, m, H-4”), 6.95–6.89 (1H, m, H-6”), 6.76 (1H, s, H-8), 6.56 (1H, s, H-6), 6.06 (1H, s, H-3), 4.21 (2H, t, J = 8 Hz, H-1′), 3.10 (4H, br. s., H-3p, H-5p), 2.95 (2H, t, J = 6 Hz, H-2′), 2.79 (4H, br. s., H-2p, H-6p), 2.62 (3H, s, H-10), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.0 (C-4), 155.5 (C-5), 154.2 (C-1”), 150.4 (C-8a), 146.9 (C-7), 143.3 (C-3”), 134.1 (C-4”), 128.6 (H-5”), 124.9 (C-6”), 121.2 (C-2”), 120.1 (C-4a), 113.9 (C-6), 110.8 (C-3), 108.6 (C-8), 66.6 (C-1′), 57.1 (C-2′), 53.9 (C-3p, C-5p), 51.5 (C-2p, C-6p), 24.8 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₃H₂₅O₃N₂BrNa (479.0946) found 479.0930.

5-(4-(4-(3-bromophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2g). Yield 69%; cream solid; m.p. 126–127 °C; Rf = 0.78; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.13–7.07 (1H, m, H-4”), 7.07–7.02 (1H, m, H-5”), 6.97–9.94 (1H, m, H-6”), 6.84–6.81 (1H, m, H-2”), 6.74 (1H, s, H-8), 6.54 (1H, s, H-6), 6.03 (1H, s, H-3), 4.18 (2H, t, J = 8 Hz, H-1′), 3.21 (4H, t, J = 6 Hz, H-3p, H-5p), 2.90 (2H, t, J = 8 Hz, H-2′), 2.71 (4H, t, J = 6 Hz, H-2p, H-6p), 2.59 (3H, s, H-10), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.1 (C-4), 154.5 (C-5), 154.3 (C-1”), 152.5 (C-8a), 143.2 (C-7), 130.7 (C-5”), 123.4 (C-3”), 122.5 (C-4”), 119.9 (C-4a), 118.9 (C-6), 115.6 (C-2”), 114.6 (C-6”), 110.5 (C-3), 108.3 (C-8), 66.7 (C-1′), 57.1 (C-2′), 53.5 (C-3p, C-5p), 48.9 (C-2p, C-6p), 24.7 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₃H₂₅O₃N₂BrNa (479.0946) found 479.0956.

5-(2-(4-(3,5-dimethylphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2h). Yield 54%; white solid; m.p. 149–150 °C; Rf = 0.90; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.78 (2H, s, H-2”, H-6”), 6.47 (2H, s H-6, H-8), 6.07 (2H, s, H-4”, H-3), 4.40–4.33 (6H, m, H-1′, H-3p, H-5p), 3.73 (4H, t, J = 8 Hz, H-2p, H-6p), 3.51 (2H, t, J = 8 Hz, H-2′), 2.64 (6H, s, H-10, H-9), 2.39 (6H, s, H-7”, H-8”); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.00 (C-2), 156.4 (C-4), 155.6 (C-5), 154.2 (C-8a), 154.0 (C-1”), 143.2 (C-7, C-3”, C-5”), 113.9 (C-4”), 111.1 (C-4a), 108.5 (C-2”), 108.4 (C-6”), 108.0 (C-6, C-3), 107.9 (C-8), 69.7 (C-1′), 68.8 (C-2′), 29.1 (C-3p, C-5p), 25.2 (C-2p, C-6p), 24.9 (C-10, C-9), 22.2 (C-7”, C-8”); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₃₀O₃N₂Na (429.2154) found 429.2165.

5-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2i). Yield 72%; ceram solid; m.p. 142–145 °C; Rf = 0.70; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.18–7.12 (2H, m, H-4”, H-3”), 6.97–6.94 (1H, m, H-2”), 6.76 (1H, s, H-8), 6.56 (1H, s, H-6), 6.05 (1H, s, H-3), 4.20 (2H, t, J = 8 Hz, H-1′), 3.09 (4H, t, br. s., H-3p, H-5p), 2.95 (2H, t, J = 8 Hz, H-2′), 2.78 (4H, br. s., H-2p, H-6p), 2.55 (3H, s, H-10), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.2 (C-4), 155.5 (C-5), 154.3 (C-8a), 151.2 (C-1”), 143.2 (C-7), 134.3 (C-3”), 127.7 (C-5”), 124.9 (C-2”), 118.8 (C-3”, C-6”), 113.8 (C-4a), 110.6 (C-6), 108.6 (C-3), 108.3 (C-8), 66.8 (C-1′), 57.2 (C-2′), 53.8 (C-3p, C-5p), 51.5 (C-2p, C-6p), 24.8 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₃H₂₄O₃N₂Cl₂Na (469.1062) found 469.1068.

5-(2-(4-(2-cyanophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (2j). Yield 74%; cream solid; m.p. 127–129 °C; Rf = 0.32; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.58–7.47 (2H, m, H-3”, H-5”), 7.05–7.01 (2H, m, H-4”, H-6”), 6.75 (1H, s, H-8), 6.56 (1H, s, H-6), 6.05 (1H, s, H-3), 4.20 (2H, t, J = 8 Hz, H-1′), 3.26 (4H, t, J = 6 Hz, H-3p, H-5p), 2.95 (2H, t, J = 8 Hz, H-2′), 2.81 (4H, t, J = 6 Hz, H-2p, H-6p), 2.62 (3H, s, H-10), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 161.1 (C-2), 157.2 (C-4), 155.7 (C-5), 155.5 (C-8a), 154.4 (C-1”), 143.2 (C-7), 134.5 (C-5”), 134.0 (C-3”), 122.1 (C-7”), 118.9 (C-4”), 118.6 (C-6”), 113.7 (C-4a), 110.5 (C-6), 108.6 (C-3), 108.3 (C-8a), 106.3 (C-2”), 66.7 (C-1′), 57.0 (C-2′), 53.6 (C-3p, C-5p), 51.7 (C-2p, C-6p), 24.7 (C-10), 22.2 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₄H₂₅O₃N₃Na (426.1794) found 426.1779.

6-acetyl-5-(5-bromopentyloxy)-4,7-dimethyl-2H-chromen-2-one (3). Yield 89%; white solid; m.p. 78–80 °C; Rf = 0.84; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.97 (1H, s, H-8), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 8.8 Hz, H-1′), 3.43 (2H, t, J = 8.8 Hz, H-5′), 2.59 (3H, s, H-12), 2.54 (3H, s, H-10), 2.29 (3H, s, H-9), 1.96–1.86 (2H, m, H-2′), 1.83–1.73 (2H, m, H-4′), 1.62–1.52 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.5 (C-11), 160.0 (C-2), 154.7 (C-5), 154.2 (C-4), 152.0 (C-8a), 139.2 (C-7), 133.5 (C-6), 116.0 (C-3), 115.2 (C-8), 112.5 (C-4a), 78.0 (C-1′), 33.3 (C-5′), 32.6 (C-12), 32.3 (C-4′), 29.0 (C-2′), 24.5 (C-3′), 22.5 (C-10), 19.3 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₁₈H₂₁O₄BrNa (403.021) found 403.0506

6-acetyl-5-(5-(4-(2-methoxyphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3a). Yield 79%; oil; Rf = 0.18; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.00–6.85 (5H, m, H-3”, H-4”, H-5”, H-6”, H-8), 6.18 (1H, s, H-3), 3.87 (3H, s, H-7”), 3.82 (2H, t, J = 6.6 Hz, H-1′), 3.11 (4H, br. s., H-3p, H-5p), 2.67 (4H, br. s., H-2p, H-6p), 2.60 (3H, s, H-12), 2.55 (3H, s, H-10), 2.44 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.84–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.49–1.41 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.7 (C-11), 161.2 (C-1”), 154.9 (C-2), 154.5 (C-4), 152.5 (C-5), 152.3 (C-8a), 141.4 (C-7), 139.4 (C-2”), 133.6 (C-6”), 123.2 (H-4”), 121.2 (C-5”), 118.4 (C-6), 116.1 (C-3), 115.3 (C-8), 112.7 (C-3”), 111.4 (C-4a), 78.6 (C-1′), 58.7 (C-3p, C-5p), 55.6 (C-5′), 53.7 (C-7”), 50.7 (C-2p, C-6p), 32.7 (C-12), 30.0 (C-2′), 26.7 (C-4′), 24.0 (C-10), 22.8 (C-3′), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₉H₃₇O₅N₂ (493.2702) found 493.2704.

6-acetyl-5-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3b). Yield 76%; cream solid; m.p. 137–139 °C; Rf = 0.32; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.08–6.90 (5H, m, H-3”, H-4”, H-5”, H-6”, H-8), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 8 Hz, H-1′), 3.13 (4H, t, J = 6 Hz, H-3p, H-5p), 2.65 (4H, t, J = 6 Hz, H-2p, H-6p), 2.60 (3H, s, H-12), 2.55 (3H, s, H-10), 2.43–2.40 (2H, m, H-5′), 2.29 (3H, s, H-9), 1.82–1.77 (2H, m, H-2′), 1.59–1.56 (2H, m, H-4′), 1.46–1.44 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 157.5 (C-5), 154.8 (C-4), 154.5 (C-2”), 154.3 (C-8a), 152.3 (C-7), 140.3 (C-1”), 139.4 (C-5”), 133.6 (C-4”), 124.6 (C-3”), 122.6 (C-6”), 119.1 (C-6), 116.4 (C-3), 155.3 (C-4a), 115.3 (C-8), 78.5 (C-1′), 58.6 (C-3p, C-5p), 53.5 (C-5′), 50.6 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.7 (C-4′), 24.0 (C-3′), 22.7 (C-10), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₄O₄N₂F (481.2503) found 481.2517.

6-acetyl-5-(5-(4-(3-methoxyphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3c). Yield 83%; white solid; m.p. 86–88 °C; Rf = 0.28; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.17 (1H, t, J = 12 Hz, H-5”), 6.97 (1H, s, H-8), 6.47 (1H, d, J = 12 Hz, H-6”), 6.46–6.40 (2H, m, H-2”, H-4”), 6.17 (1H, s, H-3), 3.84–3.79 (5H, m, H-7”, H-1′), 3.21 (4H, t, J = 5.3 Hz, H-3p, H-5p), 2.60 (7H, br. s., H-12, H-2p, H-6p), 2.55 (3H, s, H-10), 2.41 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.84–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′)1.49–1.39 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.8 (C-3”), 160.2 (C-2), 154.8 (C-4), 154.5 (C-5), 152.8 (C-8a), 152.2 (C-1”), 139.4 (C-7), 133.6 (C-5”), 129.9 (C-6), 116.0 (C-3), 115.2 (C-8), 112.7 (C-4”), 109.0 (C-4a), 104.6 (C-6”), 102.7 (C-2”), 78.5 (C-1′), 58.6 (C-3p, C-5p), 55.4 (C-7”), 53.4 (C-5′), 49.2 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.8 (C-4′), 23.9 (C-10), 22.7 (C-3′), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₉H₃₇O₅N₂ (493.2702) found 493.2713.

6-acetyl-5-(5-(4-(2,5-dimethylphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3d). Yield 75%; cream solid; m.p. 97–99 °C; Rf = 0.20; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.05 (1H, d, J = 8 Hz, H-3”), 6.97 (1H, s, H-8), 6.83–6.78 (2H, m, H-4”, H-6”), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 10 Hz, H-1′), 2.94 (4H, t, J = 6 Hz, H-3p, H-5p), 2.60 (7H, br. s, H-2p, H-6p, H-12), 2.55 (3H, s, H-10), 2.43 (2H, t, J = 10 Hz, H-5′), 2.29 (6H, s, H-9, H-7”), 2.25 (3H, s, H-8”), 1.85–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.49–1.39 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 154.9 (C-4), 154.6 (C-5), 152.3 (C-8a), 151.5 (C-1”), 139.4 (C-7), 136.2 (C-5”), 133.6 (C-2”), 131.0 (H-3”), 129.4 (C-4”), 123.9 (C-6), 119.9 (C-6”), 116.0 (C-3), 115.3 (C-8), 112.7 (C-4a), 78.6 (C-1′), 58.7 (C-5′), 54.0 (C-3p, C-5p), 51.8 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.8 (C-4′), 24.0 (C-9), 22.7 (C-3′), 21.4 (C-10), 19.5 (C-7”),17.6 (C-8”); TOF MS ES+: [M+H]⁺ calcd for C₃₀H₃₉O₄N₂ (491.2910) found 491.2898.

6-acetyl-5-(5-(4-(3-fluorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3e). Yield 68%; white solid; m.p. 150–152 °C; Rf = 0.27; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.22–7.14 (1H, m, H-5”), 6.97 (1H, s, H-8), 6.68–6.52 (3H, m, H-2”, H-4”, H-6”), 6.17 (1H, s, H-3), 3.82 (2H, t, J = 8 Hz, H-1′), 3.21 (4H, t, J = 6 Hz, H-3p, H-5p), 2.58 (7H, br. s., H-12, H-2p, H-6p), 2.55 (3H, s, H-10), 2.41 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.84–1.75 (2H, m, H-2′), 1.63–1.39 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 165.3 (C-3”), 162.4 (C-2), 160.2 (C-5), 154.8 (C-4), 153.0 (C-1”), 152.2 (C-8a), 139.3 (C-7), 133.6 (C-5”), 116.1 (C-6), 112.7 (C-3), 111.3 (C-8), 106.3 (C-4a), 106.0 (C-6”), 103.1 (C-4”), 102.7 (C-2”), 78.5 (C-1′), 58.4 (C-3p, C-5p), 53.1 (C-5′), 48.6 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.5 (C-4′), 23.9 (C-10), 22.7 (C-3′), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₄O₄N₂F (481.2503) found 481.2492.

6-acetyl-5-(5-(4-(2-bromophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3f). Yield 51.8%; cream solid; m.p. 108–109 °C; Rf = 0.31; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.55 (1H, d, J = 12 Hz, H-3”), 7.27 (1H, t, J = 12 Hz, H-5”), 7.06 (1H, d, J = 12 Hz, H-6”), 6.97 (1H, s, H-8), 6.91 (1H, t, J = 10 Hz, H-4”), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 10 Hz, H-1′), 3.09 (4H, br. s., H-3p, H-5p), 2.66 (4H, br. s., H-2p, H-6p), 2.60 (3H, s, H-12), 2.55 (3H, s, H-10), 2.44 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.85–1.75 (2H, m, H-2′), 1.64–1.42 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.1 (C-2), 154.8 (C-4), 154.5 (C-5), 152.2 (C-8a), 150.6 (C-1”), 139.4 (C-7), 133.9 (C-3”), 133.6 (C-4”), 128.5 (H-5”), 124.7 (C-6”), 121.2 (C-2”), 120.0 (C-6), 116.0 (C-3), 115.3 (C-8), 112.7 (C-4a), 78.5 (C-1′), 58.5 (C-3p, C-5p), 53.5 (C-5′), 51.5 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.5 (C-4′), 23.9 (C-3′), 22.7 (C-10), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₄O₄N₂Br (541.1702) found 541.1720.

6-acetyl-5-(5-(4-(3-bromophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3g). Yield 43%; cream solid; m.p. 100–102 °C; Rf = 0.40; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.13–6.94 (4H, m, H-2”, H-4”, H-5”, H-8), 6.83 (1H, d, J = 6 Hz, H-6”), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 10 Hz, H-1′), 3.21 (4H, t, J = 6 Hz, H-3p, H-5p), 2.60 (7H, br. s., H-12, H-2p, H-6p), 2.55 (3H, s, H-10), 2.41 (2H, t, J = 10 Hz, H-5′), 2.30 (3H, s, H-9), 1.85–1.75 (2H, m, H-2′), 1.64–1.42 (4H, m, H-3′, H-4′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 154.9 (C-4), 155.5 (C-5), 152.2 (C-1”, C-8a), 139.3 (C-7), 133.6 (C-5”), 130.5 (C-3”), 123.4 (C-4”), 122.6 (C-6), 118.9 (C-2”), 116.1 (C-6”),115.3 (C-3), 114.6 (C-8), 112.7 (C-4a), 78.4 (C-1′), 58.4 (C-3p, C-5p), 53.1 (C-5′), 48.6 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.5 (C-4′), 23.9 (C-10), 22.7 (C-3′), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₄O₄N₂Br (541.1702) found 541.1701.

6-acetyl-5-(5-(4-(3,5-dimethylphenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3h). Yield 83%; cream solid; m.p. 100–102 °C; Rf = 0.33; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.97 (1H, s, H-8), 6.56 (2H, s, H-2”, H-6”), 6.52 (1H, s, H-4”), 6.18 (1H, s, H-3), 3.81 (2H, t, J = 10 Hz, H-1′), 3.20 (4H, t, J = 10 Hz, H-3p, H-5p), 2.59 (7H, br. s, H-2p, H-6p, H-12), 2.54 (3H, s, H-10), 2.41 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 2.27 (6H, s, H-7”, H-8”), 1.84–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.48–1.41 (2H, m, H3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 154.9 (C-5), 154.5 (C-4), 152.3 (C-8a), 151.6 (C-1”), 139.4 (C-7), 138.8 (C-5”), 133.6 (C-3”), 121.9 (C-4”), 116.1 (C-6”, C-2”), 115.3 (C-6), 114.3 (C-3, C-8), 112.7 (C-4a), 78.5 (C-1′), 58.6 (C-3p, C-5p), 53.5 (C-5′), 49.4 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.8 (C-4′), 24.0 (C-10), 22.8 (C-9), 21.8 (C-3′), 19.5 (C-7”, C-8”); TOF MS ES+: [M+H]⁺ calcd for C₃₀H₃₉O₄N₂ (491.2910) found 491.2918.

6-acetyl-5-(5-(4-(2,3-dichlorophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3i). Yield 64%; white solid; m.p. 129–130 °C; Rf = 0.38; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.16–7.11 (2H, m, H-4”, H-5”), 6.97–6.95 (2H, m, H-6”, H-8), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 8 Hz, H-1′), 3.09 (4H, t, br. s., H-3p, H-5p), 2.65 (4H, br. s., H-2p, H-6p), 2.60 (3H, s, H-12), 2.55 (3H, s, H-10), 2.44 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.85–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.49–1.41 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.7 (C-11), 160.2 (C-2), 154.9 (C-5), 154.5 (C-4), 152.3 (C-8a), 139.4 (C-1”), 134.2 (C-7), 133.6 (C-3”), 127.7 (C-5”), 124.9 (C-2”), 118.8 (C-4”), 116.1 (C-6”, C-6), 115.3 (C-3, C-8), 112.7 (C-4a), 78.5 (C-1′), 58.6 (C-3p, C-5p), 53.5 (C-5′), 51.4 (C-2p, C-6p),32.7 (C-12), 29.9 (C-2′), 23.9 (C-4′), 22.8 (C-3′, C-10), 19.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₈H₃₃O₄N₂Cl₂ (531.1817) found 531.1835.

6-acetyl-5-(5-(4-(2-cyanophenyl)piperazin-1-yl)pentyloxy)-4,7-dimethyl-2H-chromen-2-one (3j). Yield 67%; brown solid; m.p. 116–118 °C; Rf = 0.38; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.58–7.46 (2H, m, H-3”, H-5”), 7.03–6.97 (3H, m, H-8, H-4”, H-6”), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 8 Hz, H-1′), 3.26 (4H, t, J = 6 Hz, H-3p, H-5p), 2.69 (4H, t, J = 6 Hz, H-2p, H-6p), 2.60 (3H, s, H-12), 2.55 (3H, s, H-10), 2.45 (2H, t, J = 10 Hz, H-5′), 2.29 (3H, s, H-9), 1.85–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.49–1.39 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 155.8 (C-4), 154.8 (C-5), 154.5 (C-8a), 152.3 (C-1”), 139.4 (C-7), 134.5 (C-5”), 134.0 (C-3”), 133.6 (C-7”), 121.9 (C-4”), 118.8 (C-6”), 118.6 (C-6), 116.1 (C-3), 115.3 (C-8), 112.7 (C-4a), 106.2 (C-2”), 78.5 (C-1′), 58.4 (C-3p, C-5p), 53.9 (C-5′), 51.6 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.7 (C-4′), 23.9 (C-10), 22.8 (C-3′), 21.5 (C-9); TOF MS ES+: [M+H]⁺ calcd for C₂₉H₃₄O₄N₃ (488.2549) found 488.2537.

6-acetyl-5-(2-bromoethoxy)-4,7-dimethyl-2H-chromen-2-one (4). Yield 50%; white solid; m.p. 168–169 °C; Rf = 0.85; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.26 (1H, s, H-8), 6.20 (1H, s, H-3), 4.15 (2H, t, J = 8 Hz, H-1′), 3.57 (2H, t, J = 8 Hz, H-2′), 2.62 (3H, s, H-12), 2.58 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.7 (C-11), 160.0 (C-2), 154.7 (C-5), 153.0 (C-3), 151.8 (C-8a), 139.4 (C-7), 133.7 (C-6), 116.5 (C-3), 115.9 (C-8), 112.7 (C-4a), 77.4 (C-1′), 33.1 (C-12), 28.9 (C-2′), 22.9 (C-10), 19.5 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₁₅H₁₅O₄BrNa (361.0051) found 361.0038.

6-acetyl-5-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4a). Yield 69%; cream solid; m.p. 132–134 °C; Rf = 0.56; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.04–6.85 (5H, m, H-3”, H-4”, H-5”, H-6”, H-8), 6.18 (1H, s, H-3), 3.96 (2H, t, J = 8 Hz, H-1′), 3.88 (3H, s, H-7”), 3.10 (4H, br. s., H-3p, H-5p), 2.76 (2H, t, J = 8 Hz, H-2′), 2.70 (4H, br. s., H-2p, H-6p), 2.66 (3H, s, H-12), 2.59 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.1 (C-1”), 154.9 (C-2, C-5), 152.4 (C-4, C-8a), 139.4 (C-7), 133.6 (C-1”), 123.4 (H-6”), 121.2 (C-4”), 118.5 (C-5”), 116.2 (C-6), 115.6 (C-3”), 112.8 (C-3), 111.5 (C-8a, C-4a), 77.6 (C-1′), 57.7 (C-2′, C-7”), 55.6 (C-3p, C-5p), 54.0 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₆H₃₀O₅N₂Na (473.2052) found 473.2059.

6-acetyl-5-(2-(4-(2-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4b). Yield 68%; cream solid; m.p. 118–119 °C; Rf = 0.84; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.10–6.91 (5H, m, H-3”, H-4”, H-5”, H-6”, H-8), 6.19 (1H, s, H-3), 3.97 (2H, br. s, H-1′), 3.13 (4H, br. s, H-3p, H-5p), 2.78 (6H, br. s, H-2′, H-2p, H-6p), 2.71–2.65 (3H, br. s, H-12), 2.61 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.8 (C-2), 160.1 (C-5), 154.8 (C-2”), 154.2 (C-4), 152.6 (C-8a), 152.3 (C-7), 139.4 (C-1”), 133.6 (C-5”), 130.0 (C-4”), 116.1 (C-3”), 115.5 (C-6”), 112.7 (C-6), 109.0 (C-3), 104.8 (C-4a), 102.7 (C-8), 75.0 (C-1′), 57.6 (C-2′), 55.4 (C-3p, C-5p), 53.8 (C-2p, C-6p), 32.9 (C-12), 22.8 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₂₇O₄N₂FNa (461.1853) found 461.1872.

6-acetyl-5-(2-(4-(3-methoxyphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4c). Yield 73%; cream solid; m.p. 75–76 °C; Rf = 0.70; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.18 (1H, t, J = 10 Hz, H-5”), 6.98 (1H, s, H-8), 6.55 (1H, d, J = 12 Hz, H-6”), 6.48–6.41 (2H, m, H-2”, H-4”), 6.18 (1H, s, H-3), 3.96 (2H, t, J = 6 Hz, H-1′), 3.80 (3H, s, H-7”), 3.21 (4H, t, J = 8 Hz, H-3p, H-5p), 2.75 (2H, t, J = 6 Hz, H-2′), 2.65–2.64 (7H, m., H-12, H-2p, H-6p), 2.60 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.1 (C-3”), 157.5 (C-2), 154.9 (C-5), 154.3 (C-4), 152.4 (C-8a), 152.3 (C-1”), 139.4 (C-7), 133.6 (C-5”), 124.7 (C-6), 119.2 (C-3), 116.5 (C-8), 116.2 (C-4”), 115.6 (C-4a), 115.5 (C-6”), 112.8 (C-2”), 76.8 (C-1′), 57.7 (C-2′), 53.9 (C-3p, C-5p), 50.5 (C-7”), 50.4 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₆H₃₀O₅N₂Na (473.2052) found 473.2067.

6-acetyl-5-(2-(4-(2,5-dimethylphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4d). Yield 75%; cream solid; m.p. 97–99 °C; Rf = 0.20; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.05 (1H, d, J = 8 Hz, H-3”), 6.97 (1H, s, H-8), 6.83–6.78 (2H, m, H-4”, H-6”), 6.18 (1H, s, H-3), 3.82 (2H, t, J = 10 Hz, H-1′), 2.94 (4H, t, J = 6 Hz, H-3p, H-5p), 2.60 (7H, br. s, H-2p, H-6p, H-12), 2.55 (3H, s, H-10), 2.43 (2H, t, J = 10 Hz, H-5′), 2.29 (6H, s, H-9, H-7”), 2.25 (3H, s, H-8”), 1.85–1.75 (2H, m, H-2′), 1.64–1.54 (2H, m, H-4′), 1.49–1.39 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.6 (C-11), 160.2 (C-2), 154.9 (C-4), 154.6 (C-5), 152.3 (C-8a), 151.5 (C-1”), 139.4 (C-7), 136.2 (C-5”), 133.6 (C-2”), 131.0 (H-3”), 129.4 (C-4”), 123.9 (C-6), 119.9 (C-6”), 116.0 (C-3), 115.3 (C-8), 112.7 (C-4a), 78.6 (C-1′), 58.7 (C-5′), 54.0 (C-3p, C-5p), 51.8 (C-2p, C-6p), 32.7 (C-12), 29.9 (C-2′), 26.8 (C-4′, 24.0 (C-9), 22.7 (C-3′), 21.4 (C-10), 19.5 (C-7”),17.6 (C-8”); TOF MS ES+: [M+H]⁺ calcd for C₃₀H₃₉O₄N₂ (491.2910) found 491.2898.

6-acetyl-5-(2-(4-(3-fluorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4e). Yield 93%; cream solid; m.p. 135–136 °C; Rf = 0.70; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.23–7.15 (1H, m, H-5”), 6.98 (1H, s, H-8), 6.69–6.65 (1H, m, H-2”), 6.62–6.50 (2H, m, H-4”, H-6”), 6.18 (1H, s, H-3), 3.96 (2H, t, J = 8 Hz, H-1′), 3.22 (4H, t, J = 8 Hz, H-3p, H-5p), 2.75 (2H, t, J = 8 Hz, H-2′), 2.67 (7H, br. s., H-12, H-2p, H-6p), 2.60 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 165.6 (C-3”), 162.4 (C-2), 160.1 (C-5), 154.8 (C-4), 154.2 (C-1”), 152.9 (C-8a), 139.5 (C-7), 133.6 (C-5”), 116.2 (C-6), 114.5 (C-4”), 112.7 (C-3), 111.3 (C-8), 111.2 (C-6”), 106.3 (C-4a), 103.0 (C-2”), 75.2 (C-1′), 57.7 (C-2′), 53.7 (C-3p, C-5p), 48.7 (C-2p, C-6p), 32.9 (C-12), 22.8 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₂₇O₄N₂FNa (461.1853) found 461.1845.

6-acetyl-5-(2-(4-(2-bromophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4f). Yield 70%; ceram solid; m.p. 83–85 °C; Rf = 0.38; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.50 (1H, dd, J₁ = 8 Hz, J₂ = 4 Hz H-3”), 7.23–7.17 (1H, m, H-5”), 6.98 (1H, dd, J₁ = 8 Hz, J₂ = 4 Hz H-4”), 6.91 (1H, s, H-8), 6.87–6.81 (1H, m, H-6”), 6.11 (1H, s, H-3), 3.90 (2H, t, J = 8 Hz, H-1′), 2.99 (4H, br. s., H-3p, H-5p), 2.71 (2H, t, J = 8 Hz, H-2′), 2.63 (4H, br. s., H-2p, H-6p), 2.59 (3H, s, H-12), 2.54 (3H, s, H-10), 2.23 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.8 (C-11), 160.2 (C-2), 154.8 (C-4), 154.4 (C-5), 152.4 (C-8a), 150.6 (C-1”), 139.5 (C-7), 134.0 (C-3”), 133.5 (C-4”), 128.5 (H-5”), 124.6 (C-6”), 121.0 (C-2”), 120.0 (C-6), 116.1 (C-3), 115.4 (C-8), 112.8 (C-4a), 75.3 (C-1′), 57.8 (C-2′), 54.0 (C-3p, C-5p), 51.7 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₂₇O₄N₂BrNa (521.1052) found 521.1048.

6-acetyl-5-(2-(4-(2-bromophenyl)piperazin-1-yl)ethgoxy)-4,7-dimethyl-2H-chromen-2-one (4g). Yield 62.4%; ceram solid; m.p. 127–128 °C; Rf = 0.31; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.10 (1H, t, J = 10 Hz, H-4”), 7.03 (1H, t, J = 4 Hz, H-5”), 6.98–6.94 (2H, m, H-6”, H-8), 6.84–6.81 (1H, m, H-2”), 6.17 (1H, s, H-3), 3.95 (2H, t, J = 8 Hz, H-1′), 3.20 (4H, t, J = 6 Hz, H-3p, H-5p), 2.74 (2H, t, J = 8 Hz, H-2′), 2.65–2.62 (7H, m, H-2p, H-6p, H-12), 2.60 (3H, s, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.1 (C-2), 154.8 (C-4), 154.1 (C-5), 152.4 (C-8a), 152.2 (C-1”), 139.4 (C-7), 133.6 (C-3”), 130.5 (C-5”), 123.4 (H-4”), 122.6 (C-6”), 118.9 (C-2”), 116.2 (C-6), 116.5 (C-3), 114.6 (C-8), 112.7 (C-4a), 74.9 (C-1′), 57.6 (C-2′), 53.6 (C-3p, C-5p), 48.6 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 19.5 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₂₇O₄N₂BrNa (521.1052) found 521.1066.

6-acetyl-5-(2-(4-(3,5-dimethylphenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4h). Yield 59%; oil; Rf = 0.42; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 6.91 (1H, s, H-8), 6.47 (2H, s, H-2”, H-6”), 6.46 (1H, s, H-4”), 6.11 (1H, s, H-3), 3.88 (2H, t, J = 6 Hz, H-1′), 3.12 (4H, t, J = 6 Hz, H-3p, H-5p), 2.67 (2H, t, J = 8 Hz, H-2′), 2.58 (7H, s, H-2p, H-6p, H-12), 2.52 (3H, s, H-10), 2.21 (6H, s, H-7”, H-8”), 2.22 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.1 (C-2), 154.9 (C-5), 154.3 (C-4), 152.4 (C-8a), 151.4 (C-1”), 139.5 (C-7), 138.9 (C-5”), 133.6 (C-3”), 122.0 (C-4”), 116.1 (C-2”), 115.5 (C-6, C-6”), 114.9 (C-3), 114.2 (C-8), 112.8 (C-4a), 75.1 (C-1′), 57.6 (C-2′), 53.9 (C-3p, C-5p), 49.3 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 21.8 (C-9), 19.6 (C-7”, C-8”); TOF MS ES+: [M+Na]⁺ calcd for C₂₇H₃₂O₄N₂Na (471.2260) found 471.2251.

6-acetyl-5-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4i). Yield 72%; yellow solid; m.p. 162–163 °C; Rf = 0.46; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.19–7.15 (2H, m, H-4”, H-5”), 6.99–6.92 (3H, m, H-6”, H-6, H-8), 6.19 (1H, s, H-3), 3.96 (2H, t, J = 8 Hz, H-1′), 3.06 (4H, t, br. s., H-3p, H-5p), 2.78 (2H, t, J = 8 Hz, H-2′), 2.70–2.66 (10H, m, H-2p, H-6p, H-12, H-10), 2.30 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.8 (C-11), 160.1 (C-2), 154.8 (C-5), 154.2 (C-4), 152.3 (C-8a), 151.1 (C-1”), 139.4 (C-7), 134.3 (C-3”), 133.5 (C-5”), 127.7 (C-2”), 124.9 (C-4”), 118.8 (C-6”), 116.2 (C-6), 115.5 (C-3, C-8), 112.7 (C-4a), 76.8 (C-1′), 57.6 (C-3p, C-5p), 53.9 (C-2p, C-6p),51.1 (C-2′), 32.9 (C-12), 22.9 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₅H₂₆O₄N₂Cl₂Na (511.1167) found 511.1147.

6-acetyl-5-(2-(4-(2-cyanophenyl)piperazin-1-yl)ethoxy)-4,7-dimethyl-2H-chromen-2-one (4j). Yield 70.5%; white solid; m.p. 153–155 °C; Rf = 0.37; ¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.59–7.47 (2H, m, H-3”, H-5”), 7.05–6.99 (3H, m, H-8, H-4”, H-6”), 6.19 (1H, s, H-3), 3.98 (2H, t, J = 6 Hz, H-1′), 3.25 (4H, br. s, H-3p, H-5p), 2.80–2.75 (6H, m, H-2p, H-6p, H-2), 2.66 (3H, s, H-12), 2.61 (3H, s, H-10), 2.31 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃, δ, ppm): 204.9 (C-11), 160.1 (C-2), 154.9 (C-4, C-5), 152.2 (C-8a, C-1”), 139.4 (C-7), 134.5 (C-5”), 134.1 (C-3”), 133.6 (C-7”), 118.9 (C-4”), 118.5 (C-6”), 116.2 (C-6, C-3), 115.6 (C-8), 112.7 (C-4a), 102.5 (C-2”), 76.8 (C-1′), 57.5 (C-2′), 53.7 (C-3p, C-5p), 51.4 (C-2p, C-6p), 32.9 (C-12), 22.9 (C-10), 19.6 (C-9); TOF MS ES+: [M+Na]⁺ calcd for C₂₆H₂₇O₄N₃Na (468.1899) found 468.1913

8-acetyl-7-(5-bromopenthoxy)-4-methylchromen-2-one (5). Yield 89%; white solid; m.p.: 101–103 °C; Rf = 0.84; ¹H NMR (400 MHz, CHCl₃) δ ppm: 7.55 (1H, d, J = 9 Hz, H-5), 6.86 (1H, d, J = 9.0 Hz, H-6), 6.14 (1H, s, H-3), 4.09 (2H, t, J = 6.2 Hz, H-1′), 3.43 (2H, t, J = 6.6 Hz, H-5′), 2.59 (3H, s, H-11), 2.39 (3H, s, H-9), 1.89 (4H, m, H-2′, H-4′), 1.63 (2H, m, H-3′); ¹³C NMR (75 MHz, CHCl₃) δ ppm: 199.4 (C-10), 160.1 (C-2), 158.0 (C-7), 152.2 (C-8a), 150.9 (C-4), 126.5 (C-5), 119.9 (C-8), 114.2 (C-6), 112.8 (C-3), 108.5 (C-4a), 69.0 (C-1′), 33.6 (C-5′), 32.6 (C-11), 32.4 (C-4′), 28.3 (C-2′), 24.8 (C-3′), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₁₇H₁₉O₄BrNa: 389.0364 found 389.0375.

8-acetyl-7-(5-[4-(2-methoxyphenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5a). Yield 83%; brown solid; m.p.: 126–128 °C; Rf = 0.22; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 8.7 Hz, H-5), 6.94 (5H, m, H-6, H-3”, H-4”, H-5”, H-6”), 6.14 (1H, s, H-3), 4.09 (2H, t, J = 8.6 Hz, H-1′), 3.86 (3H, s, H-7”), 3.14 (4H, br. s, H-3p, H-5p), 2.71 (4H, br. s, H-2p, H-6p), 2.59 (3H, s, H-11), 2.48 (2H, t, J = 7.5 Hz, H-5′), 2.39 (3H, s, H-9), 1.85 (2H, m, H-2′), 1.63 (2H, m, H-4′), 1.51 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.5 (C-10), 160.1 (C-1”), 158.1 (C-2), 152.4 (C-7), 152.3 (C-8a), 150.8 (C-4), 141.0 (C-2”), 126.5 (C-5), 123.3 (C-6”), 121.2 (C-5′), 119.8 (C-4”), 118.5 (C-8), 114.1 (C-6), 112.7 (C-3”), 111.4 (C-4), 108.5 (C-4a), 69.1 (C-1′), 58.5 (C-3p, C-5p), 55.5 (C-5′), 53.5 (C-2p), 50.2 (C-6p), 32.6 (C-11), 28.9 (C-2′, C-7”), 26.0 (C-4′), 23.9 (C-3′), 18.9 (C-9); TOF MS ES + [M + Na]⁺ calcd for C₂₈H₃₄O₅N₂Na 501.2365, found 501.2345.

8-acetyl-7-(5-[4-(2-fluorophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5b). Yield 79.6%; white solid; m.p.: 95–97 °C; Rf = 0.20; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.54 (1H, d, J = 8.7 Hz, H-5), 6.94 (5H, m, H-6, H-3”, H-4”, H-5”, H-6”), 6.13 (1H, s, H-3), 4.08 (2H, t, J = 6.3 Hz, H-1′), 3.14 (4H, br. s, H-3p, H-5p), 2.66 (4H, br. s, H-2p, H-6p), 2.58 (3H, s, H-11), 2.54 (2H, t, J = 7.5 Hz, H-5′), 2.39 (3H, s, H-9), 1.84 (2H, m, H-2′), 1.64 (2H, m, H-4′), 1.49 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.5 (C-10), 160.1 (C-2), 158.1 (C-7), 154.2 (C-2”), 152.2 (C-8a), 150.8 (C-4), 140.0 (C-1”), 126.5 (C-5), 124.7 (C-5”), 124.6 (C-4”), 122.8 (C-3”), 119.8 (C-6”), 119.2 (C-8), 116.1 (C-6), 112.7 (C-3), 108.5 (C-4a), 69.1 (C-1′), 58.4 (C-5′), 53.3 (C-3p, C-5p), 50.2 (C-2p, C-6p), 32.6 (C-11), 28.9 (C-2′), 26.2 (C-4′), 24.0 (C-3′), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₇H₃₁O₄N₂FNa 489.2166, found 489.2182.

8-acetyl-7-(5-[4-(3-methoxyphenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5c). Yield 69%; white solid; m.p.: 76–78 °C; Rf = 0.24; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.54 (1H, d, J = 8.7 Hz, H-5), 7.16 (1H, t, J = 8 Hz, H-5”), 6.86 (1H, d, J = 9 Hz, H-6), 6.55 (1H, d, J = 9 Hz, H-6”), 6.45 (1H, s, H-2”), 6.44 (1H, d, J = 8Hz, H-4”), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 6.3 Hz, H-1′), 3.78 (3H, s, H-7”), 3.22 (4H, m, H-3p, H-5p), 2.63 (4H, m, H-2p, H-6p), 2.59 (3H, s, H-11), 2.44 (2H, t, J = 7.5 Hz, H-5′), 2.39 (3H, s, H-9), 1.84 (2H, m, H-2′), 1.64 (2H, m, H-4′), 1.49 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.5 (C-10), 160.7 (C-3”), 160.1 (C-2), 158.1 (C-7), 152.6 (C-8a), 152.3 (C-4), 150.8 (C-1”), 126.9 (C-5), 126.5 (C-5”), 118.8 (C-8), 114.1 (C-6), 112.7 (C-3), 109.1 (C-4a), 108.5 (C-4”), 104.8 (C-6”), 102.8 (C-2”), 69.1 (C-1′), 58.4 (C-5′), 55.4 (C-3p, C-5p), 53.2 (C-7”), 48.9 (C-2p, C-6p), 32.6 (C-11), 28.9 (C-2′), 26.2 (C-4′), 24.0 (C-3′), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₈H₃₄O₅N₂Na 501.2365, found 501.2373.

8-acetyl-7-(5-[4-(2, 5-dimethylphenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5d). Yield 54%; white solid; m.p.: 92–94 °C; Rf = 0.17; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.55 (1H, d, J = 9 Hz, H-5), 7.05 (1H, d, J = 6 Hz, H-6), 6.84 (3H, m, H-3”, H-4”, H-6”), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 6 Hz, H-1′), 2.94 (4H, m, H-3p, H-5p), 2.59 (7H, m, H-2p, H-6p, H-11), 2.39 (5H, m, H-9, H-5′), 2.30 (3H, s, H-8”), 2.25 (3H, s, H-7”), 1.84 (2H, m, H-2′), 1.54 (4H, m, H-4′, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.1 (C-2), 158.2 (C-7), 152.2 (C-8a), 151.4 (C-4), 150.8 (C-1”), 136.2 (C-5”), 131.0 (C-2”), 129.4 (C-3”), 126.5 (C-5), 123.9 (C-4”), 119.9 (C-8), 119.8 (C-6), 114.1 (C-6”), 112.7 (C-2), 108.5 (C-4a), 69.2 (C-1′), 58.7 (C-5′), 53.9 (C-3p, C-5p), 51.8 (C-2p, C-6p), 32.6 (C-11), 29.1 (C-2′), 26.7 (C-4′), 24.1 (C-3′), 21.4 (C-8”), 18.9 (C-9), 17.6 (C-7”); TOF MS ES+: [M + Na]⁺ calcd for C₂₉H₃₆O₄N₂Na 499.2573, found 499.2560.

8-acetyl-7-(5-[4-(3-fluorophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5e). Yield 47%; white solid; m.p.: 117–119 °C; Rf = 0.24; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.55 (1H, d, J = 9 Hz, H-5), 7.17 (1H, m, H-5”), 6.86 (1H, d, J = 9 Hz, H-6), 6.60 (3H, m, H-2”, H-4”), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 7.5 Hz, H-1′), 3.20 (4H, t, J = 6 Hz, H-3p, H-5p), 2.57 (7H, m, H-2p, H-6p, H-11), 2.39 (5H, m, H-5′, H-9), 1.84 (2H, m, H-2′), 1.54 (4H, m, H-4′, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 165.6 (C-3”), 160.2 (C-2), 160.1 (C-7), 158.1 (C-8a), 153.2 (C-4), 150.8 (C-1”), 130.3 (C-5), 126.5 (C-5”), 119.8 (C-8), 114.1 (C-6), 112.7 (C-2), 112.2 (C-4a), 108.4 (C-4”), 105.8 (C-6”), 102.6 (C-2”), 69.2 (C-1′), 58.5 (C-5′), 53.3 (C-3p, C-5p), 48.8 (C-2p, C-6p), 32.6 (C-11), 29.1 (C-2′), 26.7 (C-4′), 24.1 (C-3′), 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₂₇H₃₂O₄N₂F 467.2346, found 467.2332.

8-acetyl-7-(5-[4-(2-bromophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5f). Yield 85%; cream solid; m.p.: 102–103 °C; Rf = 0.22; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.55 (2H, d, J = 9 Hz, H-5, H-3”), 7.27 (2H, m, H-6, H-5”), 7.07 (1H, d, J = 9 Hz, H-4), 6.90 (1H, m, H-6”), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 12 Hz, H-1′), 3.08 (4H, br. s, H-3p, H-5p), 2.56 (4H, br. s, H-2p, H-6p), 2.47 (3H, s, H-11), 2.45 (2H, m, H-5′), 2.42 (3H, s, H-9), 1.84 (2H, m, H-2′), 1.55 (4H, m, H-4′, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.2 (C-2), 158.2 (C-7), 152.2 (C-4), 150.8 (C-8a), 150.8 (C-1”), 133.9 (C-3”), 128.5 (C-5), 126.5 (C-4”), 124.5 (C-5”), 121.1 (C-6”), 120.0 (C-2”), 119.8 (C-8), 114.1 (C-6), 112.7 (C-2), 104.4 (C-4a), 69.2 (C-1′), 58.6 (C-5′), 53.6 (C-3p, C-5p), 51.8 (C-2p, C-6p), 32.6 (C-11), 29.1 (C-2′), 26.7 (C-4′), 24.1 (C-3′), 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₂₇H₃₂O₄N₂Br 527.1545, found 527.1537.

8-acetyl-7-(5-[4-(3-bromohenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5g). Yield 64%; white solid; m.p.: 119–121 °C; Rf = 0.19; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.54 (1H, d, J = 9 Hz, H-5), 7.09 (1H, m, H-4”), 7.02 (1H, m, H-5”), 6.94 (1H, d, J = 6 Hz, H-6), 6.88 (1H, s, H-2”), 6.83 (1H, m, H-6”), 6.14 (1H, s, H-3), 4.09 (2H, t, J = 7.5 Hz, H-1′), 3.20 (4H, m, H-3p, H-5p), 2.56 (7H, m, H-2p, H-6p, H-11), 2.39 (5H, m, H-5′, H-9), 1.85 (2H, m, H-2′), 1.56 (4H, m, H-4′, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.2 (C-2), 158.2 (C-7), 152.7 (C-8a), 152.2 (C-4), 150.9 (C-1”), 130.5 (C-5), 126.5 (C-5”), 123.4 (C-3”), 122.4 (C-4”), 119.8 (C-8), 118.8 (C-6), 114.5 (C-2”), 114.1 (C-6”), 112.8 (C-3), 108.5 (C-4a), 69.2 (C-1′), 58.5 (C-5′), 53.3 (C-3p, C-5p), 48.8 (C-2p, C-6p), 32.6 (C-11), 29.1 (C-2′), 26.7 (C-4′), 24.1 (C-3′), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₇H₃₁O₄N₂BrNa 549.1365, found 549.1378.

8-acetyl-7-(5-[4-(3, 5-dimethylphenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5h). Yield 87%; white solid; m.p.: 113–115 °C; Rf = 0.22; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.54 (1H, d, J = 9 Hz, H-6), 6.87 (1H, d, J = 9 Hz, H-5), 6.54 (3H, d, J = 12 Hz, H-2”, H-4”, H-6”), 6.14 (1H, s, H-3), 4.08 (2H, t, J = 7.5 Hz, H-1′), 3.18 (4H, t, J = 4.5 Hz, H-3p, H-5p), 2.59 (7H, m, H-2p, H-6p, H-11), 2.39 (5H, m, H-9, H-5′), 2.27 (6H, s, H-7”, H-8”), 1.84 (2H, m, H-2′), 1.55 (4H, m, H-4′, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.2 (C-2), 158.2 (C-7), 152.2 (C-8a), 151.6 (C-4), 150.9 (C-1”), 138.8 (C-5”, C-3”), 126.5 (C-5), 121.9 (C-4”), 119.8 (C-8), 114.2 (C-6), 114.1 (C-2”, C-6”), 112.8 (C-3), 108.5 (C-4a), 69.2 (C-1′), 58.7 (C-5′), 53.6 (C-3p, C-5p), 48.4 (C-2p, C-6p), 32.6 (C-11), 29.1 (C-2′), 26.7 (C-4′), 24.1 (C-3′), 21.9 (C-8”), 19.6 (C-7”), 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₂₉H₃₇O₄N₂ 477.2753, found 477.2735

8-acetyl-7-(5-[4-(2, 3-dichlorophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5i). Yield 68%; white solid; m.p.: 137–139 °C; Rf = 0.17; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.55 (1H, d, J = 8.7 Hz, H-5), 7.14 (2H, m, H-6, H-4”), 6.96 (1H, m, H-5”), 6.86 (1H, d, J = 9 Hz, H-6”), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 6.3 Hz, H-1′), 3.11 (4H, br. s, H-3p, H-5p), 2.70 (4H, br. s, H-2p, H-6p), 2.59 (3H, s, H-11), 2.49 (2H, t, J = 7.3 Hz, H-5′), 2.39 (3H, s, H-9), 1.84 (2H, m, H-2′), 1.64 (2H, m, H-4′), 1.50 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.5 (C-10), 160.1 (C-2), 158.1 (C-7), 152.2 (C-8a, C-4), 150.9 (C-1”), 134.2 (C-3”), 127.7 (C-6), 126.5 (C-5”), 125.1 (C-2”), 119.8 (C-4”), 118.9 (C-6”), 114.1 (C-8, C-6), 112.8 (C-2), 108.5 (C-4a), 69.1 (C-1′), 58.3 (C-5′), 53.3 (C-3p, C-5p), 50.8 (C-2p, C-6p), 32.6 (C-11), 28.9 (C-2′), 25.9 (C-4′), 23.9 (C-3′), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₇H₃₀O₄N₂Cl₂Na 539.1480, found 539.1464.

8-acetyl-7-(5-[4-(2-cyanophenyl)piperazin-1-yl]penthoxy)-4-methylchromen-2-one (5j). Yield 69.6%; white solid; m.p.: 59–61 °C; Rf = 0.29; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.48 (3H, m, H-5, H-3”, H-5”), 7.00 (2H, m, H-6”, H-4”), 6.86 (1H, d, J = 8.7 Hz, H-6), 6.13 (1H, s, H-3), 4.09 (2H, t, J = 6.3 Hz, H-1′), 3.27 (4H, br. s, H-3p, H-5p), 2.72 (4H, br. s, H-2p, H-6p), 2.51 (3H, s, H-11), 2.49 (2H, t, J = 7.3 Hz, H-5′), 2.39 (3H, s, H-9), 1.83 (2H, m, H-2′), 1.62 (2H, m, H-4′), 1.49 (2H, m, H-3′); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 159.9 (C-2), 157.9 (C-7), 155.3 (C-8a), 152.1 (C-4), 150.7 (C-1”), 134.3 (C-5), 133.9 (C-3”), 126.3 (C-5”), 122.1 (C-7”), 119.6 (C-4”), 118.8 (C-6”), 118.3 (C-8), 113.9 (C-6), 112.6 (C-3), 108.3 (C-4a), 106.2 (C-2”), 68.9 (C-1′), 58.0 (C-5′), 52.9 (C-3p, C-5p), 50.9 (C-2p, C-6p), 32.5 (C-11), 28.7 (C-2′), 25.7 (C-4′), 23.7 (C-3′), 18.8 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₈H₃₁O₄N₃Na 496.2212, found 496.2226.

8-acetyl-7-(2-bromoethoxy)-4-methylchromen-2-one (6). Yield 80.4 %; yellow solid; m.p.: 140–142 °C; Rf = 0.81; ¹H NMR (400 MHz, CHCl₃) δ ppm: 7.57 (1H, d, J = 8 Hz, H-5), 6.86 (1H, d, J = 12 Hz, H-6), 6.17 (1H, s, H-3), 4.40 (2H, t, J = 8 Hz, H-1′), 3.65 (2H, t, J = 8 Hz, H-2′), 2.63 (3H, s, H-11), 2.41 (3H, s, H-9); ¹³C NMR (75 MHz, CHCl₃) δ ppm: 199.2 (C-10), 159.9 (C-2), 156.9 (C-7), 152.1 (C-8a), 150.9 (C-4), 126.6 (C-5), 120.3 (C-8), 114.9 (C-6), 113.3 (C-3), 108.6 (C-4a), 69.1 (C-1′), 32.8 (C-11), 28.6 (C-2′) 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₁₄H₁₄O₄Br: 325.0075 found 325.0064.

8-acetyl-7-(2-[4-(2-methoxyphenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6a). Yield 76%; white solid; m.p.: 157–159 °C; Rf = 0.23; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-5), 6.93 (5H, m, H-6, H-3”, H-4”, H-5”, H-6”), 6.15 (1H, s, H-3), 4.26 (2H, t, J = 6 Hz, H-1′), 3.87 (3H, s, H-7”), 3.11 (4H, br. s, H-3p, H-5p), 2.90 (2H, t, J = 6 Hz, H-2′), 2.79 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 160.0 (C-1”), 157.4 (C-2), 152.4 (C-7), 152.2 (C-8a), 150.9 (C-4), 140.7 (C-2”), 126.7 (C-5), 123.6 (C-6”), 121.2 (C-5′), 119.9 (C-4”), 118.6 (C-8), 114.6 (C-6), 113.1 (C-3”), 111.5 (C-3), 108.7 (C-4a), 56.8 (C-1′, C-2′), 55.6 (C-3p, C-5p), 53.9 (C-7”), 50.0 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₂₅H₂₉O₅N₂ 437.2076, found 437.2059.

8-acetyl-7-(2-[4-(2-fluorophenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6b). Yield 53%; cream solid; m.p.: 137–139 °C; Rf = 0.15; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-5), 6.97 (5H, m, H-6, H-3”, H-4”, H-5”, H-6”), 6.15 (1H, s, H-3), 4.25 (2H, t, J = 6 Hz, H-1′), 3.12 (4H, t, J = 6 Hz, H-3p, H-5p), 2.89 (2H, t, J = 8 Hz, H-2′), 2.75 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 160.0 (C-2), 157.7 (C-7), 154.2 (C-2”), 152.2 (C-8a), 150.9 (C-4), 140.1 (C-1”), 126.6 (C-5), 124.7 (C-5′), 124.6 (C-4”), 119.9 (C-3”), 119.2 (C-6”), 116.4 (C-8), 116.2 (C-6), 112.9 (C-3), 108.6 (C-4a), 67.4 (C-1′), 56.9 (C-2′), 53.9 (C-3p, C-5p), 50.5 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + H]⁺ calcd for C₂₄H₂₅O₄N₂FNa 447.1696, found 447.1713.

8-acetyl-7-(2-[4-(3-methoxyphenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6c). Yield 93%; brown solid; m.p.: 122–124 °C; Rf = 0.33; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-5), 7.17 (1H, t, J = 6 Hz, H-5”), 6.90 (1H, d, J = 12 Hz, H-6), 6.52 (1H, m, H-6”), 6.43 (2H, m, H-3”, H-4”), 6.14 (1H, s, H-3), 4.25 (2H, t, J = 8 Hz, H-1′), 3.78 (3H, s, H-7”), 3.20 (4H, t, J = 6 Hz,, H-3p, H-5p), 2.87 (2H, t, J = 8 Hz, H-2′), 2.71 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.39 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.8 (C-3”), 160.1 (C-2), 157.7 (C-7), 152.6 (C-8a), 152.2 (C-4), 150.9 (C-1”), 130.0 (C-5), 126.6 (C-5”), 119.9 (C-8), 114.4 (C-6), 112.9 (C-3), 109.1 (C-4a), 108.6 (C-4”), 104.9 (C-6”), 102.8 (C-2”), 67.6 (C-1′), 56.9 (C-2′), 55.4 (C-3p, C-5p), 53.8 (C-2p, C-6p), 49.1 (C-7”), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₅H₂₈O₅N₂Na 459.1896, found 459.1911.

8-acetyl-7-(2-[4-(2,5-dimethylphenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6d). Yield 93%; cream solid; m.p.: 96–98 °C; Rf = 0.21; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-5), 7.05 (1H, d, J = 8 Hz, H-6), 6.91 (1H, d, J = 12 Hz H-3”), 6.79 (2H, m, H-4”, H-6”), 6.14 (1H, s, H-3), 4.26 (2H, t, J = 8 Hz, H-1′), 2.91 (6H, m, H-3p, H-5p, H-2′), 2.72 (4H, br. s, H-2p, H-6p), 2.62 (3H, s, H-11), 2.40 (3H, s, H-9), 2.30 (3H, s, H-8”), 2.25 (3H, s, H-7”); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.0 (C-2), 157.7 (C-7), 152.2 (C-8a), 151.2 (C-4), 150.9 (C-1”), 136.3 (C-5”), 131.1 (C-3”, C-5), 129.4 (C-2”), 126.6 (C-4), 124.1 (C-8), 119.9 (C-6), 114.3 (C-6”), 112.9 (C-3), 108.6 (C-4a), 67.5 (C-1′), 56.9 (C-2′), 54.3 (C-3p, C-5p), 51.7 (C-2p, C-6p), 32.6 (C-11), 21.4 (C-9), 18.9 (C-8”), 17.6 (C-7”),); TOF MS ES+: [M + Na]⁺ calcd for C₂₆H₃₀O₄N₂Na 457.2103, found 457.2116.

8-acetyl-7-(2-[4-(3-fluorophenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6e). Yield 62%; brown solid; m.p.: 86–88 °C; Rf = 0.16; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-5), 7.20 (1H, q, J = 8 Hz, H-5”), 6.90 (1H, d, J = 12 Hz, H-6), 6.60 (3H, m, H-6”, H-4”, H-2”), 6.15 (1H, s, H-3), 4.26 (2H, t, J = 8 Hz, H-1′), 3.21 (4H, t, J = 6 Hz,, H-3p, H-5p), 2.87 (2H, t, J = 8 Hz, H-2′), 2.72 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 165.6 (C-3”), 162.4 (C-2), 160.0 (C-7), 157.5 (C-8a), 152.7 (C-4), 150.9 (C-1”), 130.5 (C-5), 126.7 (C-5”), 119.9 (C-8), 114.5 (C-6), 113.1 (C-3), 111.5 (C-4a), 108.7 (C-4”), 106.6 (C-6”), 102.9 (C-2”), 67.3 (C-1′), 56.8 (C-2′), 55.5 (C-3p, C-5p), 48.5 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₄H₂₅O₄N₂FNa 447.1696, found 447.1689.

8-acetyl-7-(2-[4-(2-bromophenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6f). Yield 72%; white solid; m.p.: 145–147 °C; Rf = 0.17; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.55 (2H, m, H-5, H-3”), 7.27 (1H, m, H-6), 7.05 (1H, H-5”), 6.91 (2H, m, H-4”, H-6”), 6.15 (1H, s, H-3), 4.26 (2H, t, J = 8 Hz, H-1′), 3.07 (4H, t, J = 4 Hz, H-3p, H-5p), 2.90 (2H, t, J = 8 Hz, H-2′), 2.76 (4H, t, J = 6 Hz, H-2p, H-6p), 2.62 (3H, s, H-11), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 160.0 (C-2), 157.7 (C-7), 152.2 (C-8a), 150.9 (C-4), 150.5 (C-1”), 134.0 (C-3”), 128.5 (C-5), 126.6 (C-4”), 124.7 (C-5”), 121.1 (C-6”), 120.0 (C-2”), 119.9 (C-8), 114.4 (C-6), 112.9 (C-3), 108.6 (C-4a), 67.4 (C-1′), 56.8 (C-2′), 53.9 (C-3p, C-5p), 51.6 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₄H₂₅O₄N₂BrNa 507.0895, found 507.0876.

8-acetyl-7-(2-[4-(3-bromohenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6g). Yield 77%; yellow solid; m.p.: 110–112 °C; Rf = 0.15; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 6 Hz, H-5), 6.97 (5H, m, H-6, H-2”, H-4”, H-5”, H-6”), 6.15 (1H, s, H-3), 4.26 (2H, t, J = 6 Hz, H-1′), 3.20 (4H, t, J = 6 Hz,, H-3p, H-5p), 2.87 (2H, t, J = 8 Hz, H-2′), 2.71 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.0 (C-2), 157.6 (C-7), 152.4 (C-8a), 152.2 (C-4), 150.9 (C-1”), 130.6 (C-5), 126.6 (C-5”), 123.4 (C-3”), 122.7 (C-4”), 119.9 (C-8), 119.0 (C-6), 114.7 (C-2”), 114.5 (C-6”), 113.0 (C-3), 108.6 (C-4a), 67.4 (C-1′), 56.8 (C-2′), 53.6 (C-3p, C-5p), 48.6 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₄H₂₅O₄N₂BrNa 507.0895, found 507.0909.

8-acetyl-7-(2-[4-(3,5-dimethylphenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6h). Yield 97%; brown solid; m.p.: 120–121 °C; Rf = 0.33; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.56 (1H, d, J = 12 Hz, H-6), 6.90 (1H, d, J = 12 Hz, H-5), 6.53 (3H, d, J = 12 Hz, H-2”, H-4”, H-6”), 6.14 (1H, s, H-3), 4.25 (2H, t, J = 6 Hz, H-1′), 3.18 (4H, t, J = 8 Hz, H-3p, H-5p), 2.87 (2H, t, J = 6 Hz, H-2′), 2.71 (4H, t, J = 6 Hz, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9), 2.27 (6H, s, H-7”, H-8”); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.4 (C-10), 160.0 (C-2), 157.7 (C-7), 152.2 (C-8a), 151.3 (C-4), 150.9 (C-1”), 138.8 (C-5”, C-3”), 126.6 (C-5), 122.1 (C-4”), 119.9 (C-8), 114.4 (C-2”, C-6”), 114.3 (C-6), 112.9 (C-3), 108.6 (C-4a), 67.5 (C-1′), 56.9 (C-2′), 53.9 (C-3p, C-5p), 49.3 (C-2p, C-6p), 32.7 (C-11), 21.9 (C-8”, C-7”), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₆H₃₀O₄N₂Na 457.2103, found 457.2086.

8-acetyl-7-(2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6i). Yield 85%; white solid; m.p.: 159–161 °C; Rf = 0.23; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.57 (1H, d, J = 12 Hz, H-5), 7.14 (2H, m, H-6, H-4”), 6.94 (2H, m, H-5”, H-6”), 6.14 (1H, s, H-3), 4.26 (2H, t, J = 6 Hz, H-1′), 3.07 (4H, br. s, H-3p, H-5p), 2.90 (2H, t, J = 8 Hz, H-2′), 2.76 (4H, br. s, H-2p, H-6p), 2.61 (3H, s, H-11), 2.40 (3H, s, H-9; ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 160.0 (C-2), 157.7 (C-7), 152.2 (C-8a), 151.1 (C-4), 150.9 (C-1”), 134.2 (C-3”), 127.7 (C-5), 126.6 (C-5”), 124.9 (C-2”), 119.9 (C-4”), 118.8 (C-6”, C-8), 114.4 (C-6), 112.9 (C-3), 108.6 (C-4a), 67.4 (C-1′), 56.8 (C-2′), 53.8 (C-3p, C-5p), 51.2 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₄H₂₄O₄N₂Cl₂Na 497.1011, found 497.1026.

8-acetyl-7-(2-[4-(2-cyanophenyl)piperazin-1-yl]ethoxy)-4-methylchromen-2-one (6j). Yield 96%; cream solid; m.p.: 155–157 °C; Rf = 0.14; ¹H NMR (300 MHz, CDCl₃) δ ppm: 7.54 (3H, m, H-5, H-3”, H-5”), 7.01 (2H, m, H-6”, H-4”), 6.91 (1H, d, J = 12 Hz, H-6), 6.15 (1H, s, H-3), 4.26 (2H, t, J = 8 Hz, H-1′), 3.24 (4H, t, J = 6 Hz, H-3p, H-5p), 2.91 (2H, t, J = 8 Hz, H-2′), 2.79 (4H, t, J = 6 Hz, H-2p, H-6p), 2.62 (3H, s, H-11), 2.41 (3H, s, H-9); ¹³C NMR (75 MHz, CDCl₃) δ ppm: 199.3 (C-10), 160.0 (C-2), 157.6 (C-7), 155.5 (C-8a), 152.2 (C-4), 150.9 (C-1”), 134.5 (C-5”), 134.0 (C-3”), 126.6 (C-5), 122.2 (C-7”), 119.9 (C-4”), 118.9 (C-6”), 118.5 (C-8), 114.4 (C-6), 112.9 (C-3), 108.7 (C-4a), 106.3 (C-2”), 67.2 (C-1′), 56.7 (C-2′), 53.7 (C-3p, C-5p), 51.4 (C-2p, C-6p), 32.7 (C-11), 18.9 (C-9); TOF MS ES+: [M + Na]⁺ calcd for C₂₅H₂₅O₄N₃Na 454.1743, found 454.1744.

3.2. Docking Studies

In the computational part of this study we used a protocol similar to our previous investigation on this topic [20,21,24]. In short, the 3D models of 5HT_1A/2A receptors were prepared using homology modelling based on the crystal structures of dopamine D3 receptor (PBD code: 3PBL) and β1 adrenergic receptor (PDB code: 2Y00), respectively [34,35]. We used flexible docking algorithm as implemented in Autodock 4.2 [36] with the ligand and the following residues described in a flexible manner: D116, V117, W358, F361, F362, N386, and Y390 for 5HT_1A receptor and D155, V156, S159, W336, F339, F340, N363, and Y370 for 5HT_2A receptor. We used a 48 × 52 × 40 Å ³ box and 60 × 54 × 50 ų box for 5HT_2A receptor, centered in both cases on the binding site. We also used standard Autodock 4.2 parameters for the Lamarckian genetic algorithm, but with 100 runs for each ligand-receptor pair for a total of 132 separate runs. Schematic figures of the ligand binding sites have been prepared using the Ligand Interaction Diagram (Schrödinger Release 2020–4: Maestro, Schrödinger, LLC, New York, NY, USA, 2020).

3.3. Biological Evaluation

3.3.1. Membrane Preparation

Male Sprague–Dawley rats were decapitated, their brains removed, and placed on ice. Hippocampi were dissected and homogenized with a glass homogenizer in 30 vol. ice-cold TED buffer (50 mM Tris-HCl, 1 mM EDTA, 1mM dithiotheritol, pH 7.4). Next, the homogenate was centrifuged at 21,000× g for 30 min at 4 °C. The pellet was suspended in 30 vol TED buffer (pH 7.4) and incubated in a water bath for 10 min at 37 °C to remove endogenous serotonin. The suspension was centrifuged again at 21,000× g for 30 min at 4 °C. The pellet was resuspended in 30 vol. TED buffer (pH 7.4) and the centrifugation step was repeated. The final pellet was suspended in 10 vol 50 mM Tris-HCl (pH 7.4) and stored at −80 °C until use.

3.3.2. Antagonist Activity for the 5-HT_1A Receptor

Compounds were dissolved in 9.5% DMSO and 0.5% Kolliphor^® EL (Sigma Aldrich, Taufkirchen, Germany). Serial dilutions of the compounds tested (10⁻¹⁰–10⁻⁵ M) were incubated in triplicate with 0.8 nM [³⁵S]GTPγS in assay buffer (50 mM Tris-HCl, pH = 7.4, 1 mM EGTA, 3 mM MgCl₂, 100 mM NaCl, 30 µM GDP) and 8-OH-DPAT (final concentration 1.4 × 10⁻⁷ M) in the final assay volume of 250 µL. Hippocampal homogenates (15 μg/mL) were added to each tube as the 5-HT_1A receptor source. The final DMSO and Kolliphor^® EL concentrations were 0.95% and 0.05%, respectively. Non-specific binding was determined with 10 µM of unlabeled GTPγS. The reaction mixture was incubated for 90 min at 37 °C in a volume of 250 µL. Next, 96-well Unifilter^® Plates (Perkin Elmer, Waltham, MA, USA) were presoaked for 1 h with 50 mM Tris-HCl (pH = 7.4) before harvesting. The reaction was terminated by vacuum filtration onto filter plates with the FilterMate Harvester^® (Perkin Elmer, Waltham, MA, USA). The samples were then rapidly washed with 2 mL of 50 mM Tris-HCl (pH = 7.4) buffer. Filter plates were dried for 2 h at 50 °C. After drying, 45 µL of EcoScint-20 scintillant (Perkin Elmer) was added to every well. Radioactivity was counted in a Trilux MicroBeta² counter (Perkin Elmer). Data were analyzed with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Curves were fitted with a one-site non-linear regression model. Efficacy (Emax) and half maximal inhibitory concentration (IC50) were calculated from the Cheng–Prusoff equation and expressed as means ± SEM.

3.3.3. Membrane Preparation for the 5-HT_2A Receptor Binding

Male SD rats were decapitated and their brains removed and placed on ice. Frontal cortices were homogenized with a glass homogenizer in 30 vol ice-cold homogenization buffer (50 mM Tris-HCl. 1 mM EDTA. 5 mM MgCl₂. pH 7.4). Next, the homogenate was centrifuged at 20,000× g for 15 min at 4 °C. The pellet was suspended in 30 vol 50 mM Tris-HCl (pH 7.4) and incubated in a water bath for 15 min at 37 °C to remove endogenous serotonin. The suspension was again centrifuged at 20,000× g for 15 min at 4 °C. The pellet was resuspended in 10 vol. 50 mM Tris-HCl (pH 7.4) and the centrifugation step was repeated. The final pellet was suspended in 10 vol 50 mM Tris-HCl (pH 7.4) and stored at −80 °C.

3.3.4. 5-HT_2A Competition Binding Assay

For the 5-HT_2A assay frontal cortex homogenates (160 µg protein/mL) were incubated in triplicate with 1 nM [³H]ketanserin for 60 min at 36 °C in a 50 mM Tris-HCl (pH 7.4) buffer containing 0.1% ascorbate (3 mM CaCl₂ and 10 µM pargyline) and increasing the concentrations (10⁻⁹–10⁻⁵ M) of the compound of interest. Non-specific binding was determined in the presence of 10 μM mianserin. After incubation, the reaction mixture was deposited onto UniFilter-96 GF/B plates with the aid of a FilterMate-96 Harvester. Filter plates were presoaked beforehand with 0.4% PEI for 1 h. Next, each filter well was washed with 1.75 mL of 50 mM Tris-HCl (pH 7.4) and left to dry on a heating block set to 50 °C for 2 h. Then 45 µL of Microscint-20 scintillation fluid was added to each filter well and left to equilibrate overnight. Filter-bound radioactivity was counted in a MicroBeta² Microplate Counter. Binding curves were fitted with one site non-linear regression. Affinity was presented as the inhibitory constant (pK_i ± SEM and K_i ± SEM) from two or three separate experiments.

4. Conclusions

Sixty new aryl-piperazinyl derivatives of 5-hydroxy-4,7-dimethylchromen-2-one (A), 6-acetyl-5-hydroxy-4,7-dimethylchromen-2-one (B) and 8-acetyl-7-hydroxy-4-methylchromen-2-one (C) were designed, synthesized, and evaluated in silico and experimentally for their 5-HT_1A and 5-HT_2A receptor-binding affinities. Figure 3 present summary of the results for the most active compounds. Five compounds showed high antagonistic activities against the 5-HT_1A receptor (1a, 3a, 4a, 5a, and 5b), though lower than WAY-100635, the reference 5HT_1A antagonist, while three compounds showed moderate affinity for 5-HT_2A receptors (5i, 1j, and 5g) with respect to ketanserin. The designed derivatives had two- or five-carbon alkyl linkers between coumarin and arylpiperazinyl moiety. The studies showed that the new compounds showed less profound binding for the tested serotonin receptors than the derivatives containing three- or four-carbon linkers, which we described in our previous works. While the differences in 5HT_1A activities between the three-carbon or four-carbon linker derivatives were minimal, further shortening or lengthening of the linker quite significantly lowered the potency of coumarin derivative to bind to this receptor. Overall, the results for the series of 5- and 7-hydroxycoumarin derivatives obtained in this and our previous investigations on this topic provide an exhaustive structure–activity relationship database, which can be used in future search for novel agents acting on serotonin receptors, either based on coumarin derivatives or other organic scaffolds.