Synthesis, Docking, 3-D-Qsar, and Biological Assays of Novel Indole Derivatives Targeting Serotonin Transporter, Dopamine D2 Receptor, and Mao-A Enzyme: In the Pursuit for Potential Multitarget Directed Ligands

Abstract

A series of 27 compounds of general structure 2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-{4-[3-(1H-3indolyl)-propyl]-1-piperazinyl}-ethanamides, Series I: 7(a–o) and (2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamine)-N-(2-morfolin-4-yl-ethyl)-fluorinated benzamides Series II: 13(a–l) were synthesized and evaluated as novel multitarget ligands towards dopamine D₂ receptor, serotonin transporter (SERT), and monoamine oxidase-A (MAO-A) directed to the management of major depressive disorder (MDD). All the assayed compounds showed affinity for SERT in the nanomolar range, with five of them displaying Ki values from 5 to 10 nM. Compounds 7k, Ki = 5.63 ± 0.82 nM, and 13c, Ki = 6.85 ± 0.19 nM, showed the highest potencies. The affinities for D₂ ranged from micro to nanomolar, while MAO-A inhibition was more discrete. Nevertheless, compounds 7m and 7n showed affinities for the D₂ receptor in the nanomolar range (7n: Ki = 307 ± 6 nM and 7m: Ki = 593 ± 62 nM). Compound 7n was the only derivative displaying comparable affinities for SERT and D₂ receptor (D₂/SERT ratio = 3.6) and could be considered as a multitarget lead for further optimization. In addition, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds in the most relevant protein targets were carried out. Furthermore, in order to obtain information on the structure–activity relationship of the synthesized series, a 3-D-QSAR CoMFA and CoMSIA study was conducted and validated internally and externally (q² = 0.625, 0.523 for CoMFA and CoMSIA and r²_ncv = 0.967, 0.959 for CoMFA and CoMSIA, respectively).

1. Introduction

Major depressive disorder (MDD) is a common, chronic, recurring, heterogeneous, and potentially life-threatening disease affecting up to 20% of the world population, according to World Health Organization [1]. MDD is triggered by a complex pattern of genetic, epigenetic, and environmental factors and, despite its prevalence, it is not being properly treated [2,3,4]. As a result, such a devastating disorder is enormously costing, economically, socially, and individually [5,6].

Since the monoamine hypothesis of depression was developed in the 1950s [7], antidepressant drug discovery has been a very active research field [8], although MDD’s underlying mechanism and neurological basis are not yet fully understood [9,10,11]. The global antidepressants market is expected to grow from $14.3 billion in 2019 to about $28.6 billion in 2020 as mental health issues are expected to surge because of the Covid-19 pandemic making an impact on the global economy.

Thus far, the vast majority of marketed antidepressants act through the serotoninergic and norepinephrinergic systems, elevating synaptic levels of the corresponding neurotransmitters by blocking the monoamine transporters [12,13,14,15]. Many adverse effects associated with first-generation antidepressants (mainly tricyclic and monoamine oxidase inhibitors, –MAOi–) were partially overcome by the arrival of second-generation antidepressants, including selective serotonin reuptake inhibitors (SSRIs), norepinephrine reuptake inhibitors (NRIs), and dual antidepressants (SNRIs), with the latter being advantageous as they can treat a wide range of symptoms [16,17,18,19]. However, much less approved antidepressants do target the dopamine system, notwithstanding dopamine has also been implicated in the pathophysiology of depression [20].

Inside the central nervous system (CNS), dopamine plays pivotal roles in executive functions, motor control, emotion, cognition, reinforcement, and reward, among others [21,22,23]. Its dysfunction is related to the pathophysiology of several CNS disorders, such as Parkinson’s disease, multiple sclerosis, schizophrenia, drug addiction, and MDD [24,25,26]. Even though depression has been historically associated with misregulation within the serotonin/norepinephrine system, in the last years, an increasing number of voices have been raised supporting that dopamine system dysfunction strings along with it [24,27,28].

Due to the aforementioned causes, MDD is nowadays recognized as a complex dynamic system [29] endowed with a complex pathophysiology. For this reason, depression symptoms and its underlying causes are more likely to be treated by a multitarget approach, by using promiscuous drugs also defined as multi-target directed ligands (MTDLs): A single drug molecule that selectively targets multiple receptors, so that different pathways conducting to the disease can be modified by using a single molecule [20,30,31,32].

Indole derivatives have always been recognized as serotoninergic modulators, given their related structural connection. Furthermore, in our research group, we have extensive experience in the synthesis of indolylpropyl-piperazine derivatives, which have been successfully employed by us [33,34,35,36,37] and other groups [38,39] as very potent serotonin transporter (SERT) ligands. On the other hand, molecular structures containing morpholine [40] or benzoxazinone [41,42,43,44,45] cores have been reported as MAOi or dopamine D₂ receptor modulators, respectively. In the light of such a background, we decided to conduct an exploratory study by merging the aforementioned indolylpropyl-piperazines and morpholine/benzoxazinone units. Herein, we describe the synthesis, docking, QSAR, and biological evaluation of two series of molecular hybrids targeting SERT, MAO-A, and D₂ receptor in the pursuit of promising multi-target leads for potential MDD treatment.

2. Results and Discussion

2.1. Chemistry Series I

2,3-Dihydro-benzo[b][1,4] oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide derivatives 7a–o

The synthetic approach for the preparation of the target compounds 7a–o is outlined below. The reaction took place between piperazine benzoxazine derivatives 6(a–c) obtained in a six-step sequence and 82–95% yields (Scheme 1), with 3-indolyl tosylates 1a–c (R = H, F, Br) obtained by reported literature procedures [37,46] to give nine final compounds 7(a, b, c, g, h, i, m, n, o) in a 42–89% yield (Scheme 2).

The synthesis of the derivatives 7d–f and 7j–l was accomplished by a different synthetic strategy, involving indolylpiperazines 9a–b [35], which were reacted with N-chloroacetyl benzoxazines 4d–f and 4j–l to give the aforementioned six final compounds (7d–f and 7j–l) with good to excellent yields (Scheme 3).

In summary, 15 compounds were synthesized for Series I in yields ranging from 42% to 91%.

2.2. Chemistry Series II

(2-{4-[3-(1H-Indol-3-yl)-propyl]-1-piperazinyl}-acetylamine)-N-(2-morpholin-4-yl-ethyl)-fluorinated benzamides

The synthetic pathway of this series involved the fluorinated benzamide derivatives 12a–d, which were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence with good to excellent yields as shown in Scheme 4.

The fluorinated benzamides derivatives 12a–d were finally connected to indolylpropylpiperazines 9a–c to achieve the expected compounds 13a–l, with yields ranging from 47% to 85% (Scheme 5).

2.3. Pharmacology Series I

Table 1. Affinities, measured as Ki values at the serotonin transporter (SERT), D₂ receptor, and percent of monoamine oxidase-A (MAO-A) inhibition (at 100 µM) of indolepiperazinyl benzoxazine derivatives (Series I).

Compound	R	R1	R2	SERT (nM)	D2 (nM)	% Inhibition MAO-A at 100 µM
Fluoxetine	-	-	-	3.15 ± 0.32	N.D.	N.D.
Haloperidol	-	-	-	N.D.	3.88 ± 0.44	N.D.
7a	-H	-H	-H	307.10 ± 30.46	4840 ± 68	27%
7b	-H	-H	-F	180.80 ± 19.97	4740 ± 131	43%
7c	-H	-F	-H	68.78 ± 1.02	2387 ± 163	40%
7d	-H	-Cl	-H	81.39 ± 4.97	1265 ± 182	18%
7e	-H	-Br	-H	36.57 ± 0.55	910 ± 263	38%
7f	-H	-OCH3	-H	412.20 ± 57.86	4407 ± 647	58%
7g	-F	-H	-H	9.63 ± 0.31	2315 ± 64	41%
7h	-F	-H	-F	7.61 ± 0.02	2317 ± 163	0%
7i	-F	-F	-H	7.51 ± 0.12	1681 ± 79	0%
7j	-F	-Cl	-H	12.30 ± 2.31	902 ± 4	20%
7k	-F	-Br	-H	5.63 ± 0.82	1041 ± 232	20%
7l	-F	-OCH3	-H	12.60 ± 1.52	3322 ± 256	52%
7m	-Br	-H	-H	50.18 ± 2.47	593 ± 62	30%
7n	-Br	-H	-F	84.44 ± 2.03	307 ± 6	23%
7o	-Br	-F	-H	34.89 ± 0,32	1152 ± 227	26%

Data represent the mean ± SEM of three experiments, each with triplicate samples. All Ki values are in nM. N.D. not determined.

summarizes the affinity of compounds 7a–o for SERT, D₂ receptor, and MAO-A. Most compounds were potent and clearly selective as SERT ligands, showing in all cases affinities in the nanomolar range, whereas the affinities for D₂ and MAO-A ranged from micromolar to much higher values, respectively. A detailed analysis of SERT activities indicates that a C-5 substitution of the indole ring with halogens (fluorine or bromine; compounds 7g or 7m) leads to more potent compounds than the unsubstituted derivative (7a). On the other hand, the presence of a halogen atom at C-6 of the benzoxazine ring increased the affinity (e.g., 7c, 7d, and 7e vs. 7a). Accordingly, the most potent compounds were those exhibiting a dual halogen substitution pattern (7i, 7j, and 7k), with Ki values below 10 nM. The C-7 halogen substitution on the benzoxazine ring gave no consistent effects, slight increases (7b and 7h) or decreases (7n) of affinity were observed, as compared with the corresponding C-7 unsubstituted compounds (7a, 7g, and 7m, respectively). The D₂ receptor affinity for this series indicates that no conclusive structure–activity relationships can be extracted for these compounds. Nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen atom at the C-5 of the indole ring and the other at either the C-6 or C-7 of the benzoxazine moiety (7h–7k, 7m–7o), resulted in more potent compounds than their corresponding monohalogenated or unsubstituted counterparts (7a–7e, 7g). Moreover, the presence of a methoxyl group at the C-6 of the benzoxazine ring has almost no effect on the affinity of the compounds for D₂ receptor. It is worth mentioning that the dihalogenated compound 7n was the only derivative displaying comparable affinities for SERT and D₂ receptor (D₂/SERT ratio = 3.6) and could be considered as a potential leader in the search of more potent multitarget compounds.

2.4. Docking Simulation Series I

Considering the pharmacological results, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds were carried out only in the human SERT (hSERT) and in selected cases at the D₂ receptor.

2.4.1. hSERT

The most potent compounds 7g, 7h, 7i, and 7k showed a common docking pose (Figure 1), which favors the following stabilizing interactions: A π–π interaction between the indole ring and the π–donor aromatic residue Tyr176, a coulombic interaction between the protonated piperazine N-1 with the Asp98 residue, and a π–cation interaction for the protonated piperazine with Tyr95. Furthermore, aromatic interactions were also observed for the benzoxazine ring with the residues Phe341 and Phe335. These drug–target interactions are in agreement with those described in the crystal structure of the hSERT in complex with the inhibitor (S)-citalopram [47,48].

The relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds 7f and 7l. Both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for 7f. Thus, the least potent compound of the series (7f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the C-5 fluorinated counterpart (Figure 2). Compounds showing intermediate affinities (7a–7e and 7m–7o) exhibited docking poses between the most and least favorable binding modes (not shown).

2.4.2. D₂ Receptor

Docking simulations showed that compounds of this series adopt, at the D₂ receptor, a binding mode similar to that experimentally determined for the atypical antipsychotic risperidone [49]. Thus, the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined by Cys118, Thr119, Ser197, Phe198, and Trp386, while the protonated piperazine N-1 locates in a favorable position to establish a coulombic interaction with Asp114 (Figure 3). Furthermore, the benzoxazine portion extends to the additional hydrophobic sub-pocket lined by Val91, Trp100, Phe110, and Tyr408, in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety of risperidone. Interestingly, this general binding mode was observed for both the most and the least potent compounds of this series (7a, 7b, and 7m, 7n), respectively (Figure 3A,B). Therefore, it is tempting to speculate that the higher affinity showed by brominated derivatives (7m and 7n) is due to the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue (e.g., Ser197). As observed (Figure 3), this could also change the position of the benzoxazine moiety, favoring its interactions at the more external hydrophobic sub-pocket.

2.5. Pharmacology Series II

Table 2. Affinities, measured as Ki values at SERT, D₂ receptor, and percent of MAO-A inhibition (at 100 µM) of indolepiperazinylmorpholinoethyl benzamide derivatives (Series II).

Compound	R	Position (Indolylpropyl Piperazineacetamide)	Position F	SERT (nM)	D2 (nM)	% Inhibition MAO-A at 100 µM
Fluoxetine	-	-	-	3.15 ± 0.32	N.D.	N.D.
Haloperidol	-	-	-	N.D.	3.88 ± 0.44	N.D.
13a	F	2	4	16.04 ± 0.21	5455 ± 25	19%
13b	F	3	4	23.78 ± 0.46	3100 ± 42	42%
13c	F	5	2	6.83 ± 0.19	592.60 ± 30.22	28%
13d	F	2	5	98.14 ± 1.24	9060 ± 29	30%
13e	Br	2	4	222.60 ± 16.44	5619 ± 31	39%
13f	Br	3	4	58.85 ± 1.14	1832 ± 33	39%
13g	Br	5	2	12.62 ± 0.42	765 ± 16	16%
13h	Br	2	5	199.60 ± 14.53	4700 ± 168	9.4%
13i	H	2	4	9945 ± 383	48910 ± 1279	23%
13j	H	3	4	109.90 ± 3.26	2339 ± 45	44%
13k	H	5	2	26.81 ± 0.91	4169 ± 335	34%
13l	H	2	5	52.16 ± 3.24	2000 ± 34	34%

Data represent the mean ± SEM of three experiments, each with triplicate samples. All Ki values are in nM range. N.D. no determined.

summarizes the affinity of Series II compounds 13a–13l for SERT, D₂ receptor, and MAO-A. As in the case of indole benzoxazine derivatives (Series I), most indole morpholine ethylbenzamides (Series II) were potent SERT ligands, showing much lower affinities for D₂ receptor and virtually no effect upon MAO-A activity. Regarding SERT activity, and in agreement with our previous studies, halogen substitution at C-5 of the indole ring with fluorine or bromine (compounds 13a–g) conducted an increase in affinity as compared with the unsubstituted analogues 13i–l, with the fluoro derivatives 13a–d being the most potent of the series. On the other hand, when the acetanilide portion, connected to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at C-2) and a morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was located at meta position (compounds 13c, 13g, and 13k).

2.6. Docking Simulation Series II

Similar to the analysis of Series I and considering the pharmacological results, docking studies were carried out only in hSERT. In this series, seven compounds exhibited Ki values between 7 and 60 nM (13a, 13b, 13c, 13f, 13g, 13k, and 13l). Docking simulations showed that compounds with the lowest Ki values (13c and 13g) share a common binding mode into the S1 site of the SERT, which is similar to that described for compounds of Series I (Figure 4A). Thus, the piperazine N-1 can establish ionic and π–cation interactions with Asp98 and Tyr176, respectively, while the indole moiety can participate in aromatic interactions with Tyr176 and Phe341. Interestingly, the ethylmorpholinic chain extends towards the extracellular vestibule (also known as the S2 site). On the other hand, for the compounds with the lowest affinities (13e and 13i), docking simulations showed that the piperazine N-1 was located farther away from Asp98 and Tyr95, making the possible ionic interactions with these residues unlikely or much weaker (Figure 4B). The analysis of the docking poses indicates that the most potent compounds, i.e., those having a 5,2-substitution pattern (13c, 13g, and 13k) exhibited an extended conformation at the binding site, while the least potent compounds (13e and 13i, showing a 2,4-substitution pattern) adopted a more constrained binding mode, impairing the most relevant interactions.

2.7. 3-D-QSAR Study

To systematize the structure–activity relationship of the synthesized molecules, we carried out a 3-D-QSAR study of the CoMFA and CoMSIA type. The complete series of 27 molecules was divided into training (19 compounds) and test sets (8 compounds) in a ratio of 70:30, selecting the test set compounds at random to avoid bias. The q² values for the best models were 0.625 and 0.523 for CoMFA and CoMSIA, respectively while the r²_ncv values were 0.967 and 0.959 for CoMFA and CoMSIA, respectively. The statistical summary, as well as the tables of affinities for both models and their respective graphs, are incorporated in the Supplementary Material.

The steric contour map of CoMFA (Figure 5A) shows a green polyhedron on the bromine atom of compound 7k, the most active of the series. This means that the insertion of bulky atoms or groups in this position is favorable for biological activity. This is consistent with docking studies showing that compounds of series I place halogen into the void space close to lipophilic residues like Trp100 and Tyr408. In the case of compounds of series II, the meta-substituted benzamides placed the chain towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the meta-position. This is confirmed in the docking of these compounds, in which better accommodation is observed in the SERT binding site. On the other hand, the electrostatic contour map (Figure 5B) shows three blue polyhedra of significant size. This means that the presence of positively charged atoms in these positions would be favorable for affinity. Such polyhedra are located on the carbon atom bonded to the halogen in the case of series I, suggesting that the presence of electronegative atoms bonded to the aforementioned carbon is favorable. The second blue polyhedron is localized on the oxygen atom of the carbonyl group belonging to the ortho-substituted series II amide-compounds. Therefore, oxygen atom remotion would be favorable for affinity. Finally, the third polyhedron is observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series II, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better affinities. Furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted in less favorable affinities compared to meta substitutions as was experimentally corroborated.

The hydrophobic contour map of CoMSIA (Figure 5C) showed a gray polyhedron at position C5 of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. In fact, the C5 fluorine-substituted indoles displayed the best affinities of the series. Other polar groups like OH, NH₂, or NR₂ would also be interesting to evaluate at this position. Similarly, a yellow polyhedron located on the bromine atom of the benzoxazine framework (compound 7k) means that the presence of lipophilic groups is favorable for activity. In concordance, halogens like Cl, Br, and I would be the most appropriated substituents and groups, such as aromatic rings, alkyl, and/or alkoxy chains, could also be explored. In the case of compounds of series II, a yellow polyhedron is located on the amide group of the meta-substituted compounds; therefore, the replacement of the amide by a less-polar function, such as a ketone or ester, would be an interesting option to explore. On the other hand, the electrostatic contour map of CoMSIA (Figure 5D) showed two polyhedra around compound 7k. A red polyhedron on the halogen atom at position 5 of the indole ring means that the presence of electron-rich atoms is favorable for affinity. It is interesting to note that the blue polyhedron intersecting the carbon atom of indole at position 5 is complementary to the red polyhedron. In consequence, the presence of a positive charge on the indole ring is favorable for activity. Other potential electron-withdrawing groups to be explored are CN, NO₂, and COR. Docking studies showed π–stacking interaction between the π-deficient indole ring with Tyr176, Phe341, and Trp386 residues.

3. Materials and Methods

3.1. General Methods

Melting points were determined on a hot-stage apparatus and were uncorrected. The ¹H and ¹³C-NMR spectra were obtained on a Bruker DRX-300 spectrometer (300 and 75 MHz, respectively) in CDCl₃, DMSO-d₆, and CD₃COCD₃-d₆. Chemical shifts were recorded in ppm (δ) relative to TMS as an internal standard. J values are given in Hz. Micro-analyses were carried out on a Fisons EA 1108 analyzer. High-resolution mass spectra were recorded on a DSA–TOFAxION 2 TOF MS (Perkin Elmer, Shelton, CT, USA), positive mode. Silica gel Merck 60 (70–230 mesh) and aluminum sheets coated with silica gel 60 F254 were used for column and TLC chromatography, respectively.

3.1.1. General Procedure for the Synthesis of [4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-piperazine-1-yl] tert-butylcarbamate Derivatives 5a–c

[4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate (5a) as a Model

To a solution containing 2-Chloro-1-(2,3-dihydrobenzo[b][1,4]oxazin-4-yl) ethanamide 4a (1.5 g; 7.09 mmol) in dry CH₃CN (60 mL), N-Boc-piperazine (1321 mg; 7.09 mmol) and anhydrous K₂CO₃ (980 mg; 7.09 mmol) were added. The mixture was stirred at 80 °C for 24 h. After this time, the mixture was diluted with water (100 mL) and the solution extracted with EtOAc (100 mL × 3), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The organic crude was purified by silica gel column chromatography with EtOAc as eluent, to provide 5a (2152 mg; 84% yield) as a white solid. m.p.: 133.0–134.0 °C; ¹H-NMR (CDCl₃): δ 1.45 (s, 9H, H-4′), 2.52 (t, 4H, H-3′,J = 4.8 Hz), 3.39 (s, 2H, H-1′), 3.45 (t, 4H, H-2′, J = 4.5 Hz), 3.99 (t, 2H, H-3, J = 4.8 Hz), 4.31 (t, 2H, H-2, J = 4.4 Hz), 6.91 (m, 2H, H-6 and H-8), 7.08 (t, 1H, H-7, J = 7.3 Hz), and 7.99 (br. s, 1H, H-5) ppm. ¹³C-NMR (CDCl₃): δ 27.9 (3X), 42.6, 43.2 (2X), 52.4 (2X), 55.4, 66.4, 79.3, 116.8, 119.7, 123.5, 125.6, 146.3, 154.2, 167.3, and 167.5 ppm. HRMS: (EI) Calculated for C₁₉H₂₇N₃O₄ (M⁺) = 362.20799. Found: 362.2113.

[4-[2-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate (5b)

2-Chloro-1-(7-fluoro-2,3-dihidro-benzo [1,4]oxazin-4-yl)-ethanamide 4b (1.5 g; 6.53 mmol), N-Boc-piperazine (1216 mg; 6.53 mmol), and anhydrous K₂CO₃ (902 mg; 6.53 mmol), to afford 5b (1983 mg; 80% yield) as a white solid. m.p.: 126.6–128.5 °C; ¹H-NMR (CDCl₃): δ 1.46 (s, 9H, H-4′), 2.51 (t, 4H, H-2′, J = 4.9 Hz), 3.35 (s, 2H, H-1′), 3.45 (t, 4H, H-3′, J = 4.8 Hz), 3.97 (t, 2H, H-3, J = 4.8 Hz), 4.30 (t, 2H, H-2, J = 4.1 Hz), 6.58–6.66 (m, 2H, H-6, and H-8), and 8.03 (br. s, 1H, H-5) ppm. ¹³C-NMR (CDCl₃): δ 27.9 (3X), 42.5 43.2 (2X), 52.4 (2X), 62.1, 66.4, 79.3, 103.8 (d, ²J_C-F = 27 Hz), 106.7 (d, ^2′J_C-F = 22.5 Hz), 121.8 (d, ³J_C-F = 9.3 Hz), 124.6 (d, ⁴J_C-F = 8.2 Hz), 147.3 (d, ^3′J_C-F = 6 Hz), 154.2, 155.6 (d, ¹J_C-F = 222 Hz), and 167 ppm. HRMS: (EI) Calculated for C₁₉H₂₆FN₃O₄ (M⁺) = 380.19856. Found: 380.2047.

[4-[2-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate (5c)

2-Chloro-1-(6-fluoro-2,3-dihidro-benzo [1,4]oxazin-4-yl)-ethanamide 4c (1.5 g; 6.53 mmol), N-Boc-piperazine (1216 mg; 6.53 mmol), and anhydrous K₂CO₃ (902 mg; 6.53 mmol), to afford 5c (2033 mg; 82% yield) as a white solid. m.p.: 113.4–114.5 °C; ¹H-NMR (CDCl₃): δ 1.46 (s, 9H, H-4′), 2.53 (t, 4H, H-2′, J = 4.8 Hz), 3.35 (s, 2H, H-1′), 3.46 (t, 4H, H-3′, J = 4.8 Hz), 3.98 (t, 2H, H-3, J = 4.9 Hz), 4.27 (t, 2H, H-2, J = 4.4 Hz), 6.77–6.87 (m, 2H, H-6 and H-8), and 7.82 (br. s, 1H, H-5) ppm. ¹³C-NMR (CDCl₃): δ 27.9 (3X), 42.5 43.4 (2X), 52.3 (2X), 62.1, 65.6, 79.3, 110.1 (d, ²J_C-F = 28.5 Hz), 112.2 (d, ^2′J_C-F = 23.6 Hz), 117.1 (d, ³J_C-F = 9.3 Hz), 125.7 (d, ⁴J_C-F = 4.9 Hz), 142.2 (d, ^3′J_C-F = 9.3 Hz),154.2, 155.5 (d, ¹J_C-F = 238 Hz), and 167.1 ppm. HRMS: (EI) Calculated for C₁₉H₂₆FN₃O₄ (M⁺) = 380.19856. Found: 380.2043.

3.1.2. General Procedure for the Synthesis of 1-(2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-piperazin-1-yl-ethanamide Derivatives 6a–c

Synthesis of 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6a) as a Model

A mixture of [4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate 5a (2 g; 5.53 mmol) in dry CH₂Cl₂ (20 mL) and trifluoroacetic acid (12 mL) was stirred at 0 °C, for 4 h. After this time, dry CH₂Cl₂ (200 mL) was added and neutralized with solid NaHCO₃ (10 g) to later filter on celite. The mixture was finally diluted with a saturated solution of NaHCO₃ (200 mL), extracted with EtOAc (8 × 50 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum to obtain pure 6a (867 mg; 82% yield) as an unstable yellow light solid, highly hygroscopic; ¹H-NMR (DMSO-d₆): δ 2.37 (m, 4H, H-2′), 2.69 (m, 4H, H-3′), 3.29 (s, 2H, H-1′), 3.89 (t, 2H, H-3, J = 4.4 Hz), 4.25 (t, 2H, H-2, J = 4.1 Hz), 6.85 (m, 2H, H-6 and H-8), 7.0 (t, 1H, H-7, J = 7.6 Hz) and 7.85 (br. s, 1H, H-5) ppm. ¹³C-NMR (DMSO-d₆): δ40.5, 45.2 (2X), 53.2 (2X), 62.0, 66.1, 73.5, 116.7, 119.6, 123.8, 125.1, 146.2, and 167.8 ppm.

1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6b)

[4-[2-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]tertbutyl carbamate 5b (2 g; 5.27 mmol) and trifluoroacetic acid (12 mL), in dry CH₂Cl₂ (20 mL) reacted to provide 6b (1398 mg; 95% yield) as a brown light hygroscopic compound; ¹H-NMR (CDCl₃): δ 2.5 7 (m, 4H, H-2′), 2.94 (t, 4H, H-3′, J = 4.4 Hz), 3.18 (br. s, 1H, H-4′), 3.33 (s, 2H, H-1′), 3.97 (t, 2H, H-3, J = 4.8 Hz), 4.31 (t, 2H, H-2, J = 4.2 Hz), 6.59–6.65 (m, 2H, H-6, and H-8), and 8.0 (br. s, 1H, H-5) ppm. ¹³C-NMR (CDCl₃): δ 45.6 (2X), 53.8 (2X), 60.4, 66.9, 77.3, 104.2 (d, ²J_C-F = 24 Hz), 107.2 (d, ^2′J_C-F = 23 Hz), 114.5 (d, ⁴J_C-F = 7 Hz), 125.1 (d, ³J_C-F = 9.4 Hz), 143.4 (d, ^3′J_C-F = 15 Hz), 155.7 (d, ¹J_C-F = 250 Hz), and 171.2 ppm.

1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6c)

[4-[2-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]tertbutyl carbamate 5c (2 g; 5.27 mmol) and trifluoroacetic acid (12 mL), in dry CH₂Cl₂ (20 mL) reacted to provide 6c (1369 mg; 93% yield) as a brown light hygroscopic compound; ¹H-NMR (CDCl₃): δ 2.62 (m, 4H, H-2′), 2.99 (t, 4H, H-3′, J = 4.6 Hz), 3.35 (s, 2H, H-1′), 3.97 (m, 3H, H-3 and H-4′), 4.28 (t, 2H, H-2, J = 4.5 Hz), 6.76–6.87 (m, 2H, H-7 and H-8) and 7.71 (br. s, 1H, H-5) ppm; ¹³C-NMR (CDCl₃): δ 45.6 (2X), 53.8 (2X), 60.4, 66.9, 77.2, 110.6 (d, ²J_C-F = 28 Hz), 112.8 (d, ^2′J_C-F = 32 Hz), 117.6 (d, ³J_C-F = 9.4 Hz), 126.1 (d, ^3′J_C-F = 11 Hz), 142.8 (d, ⁴J_C-F = 6.6 Hz), 157.6(d, ¹J_C-F = 237 Hz), and 171.2 ppm.

3.1.3. General Procedure for the Synthesis of 2,3-dihydro-benzo[b][1,4] oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide Derivatives 7a–c, 7g–i, and 7m–o. Method 1

1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7a) as a Model

To a stirred solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (254 mg. 0.77 mmol), in dry CH₃CN (50 mL), benzoxazine 6a (200 mg, 0.77 mmol) and anhydrous K₂CO₃ (106 mg; 0.77 mmol) were added. The mixture was heated at 80 °C for 24 h. After this time, the resulting mixture was poured into water (100 mL), extracted with EtOAc (4 × 50 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The organic crude was purified by column chromatography EtOAc/MeOH (6:1) to give compound 7a (217 mg; 70% yield) as a yellow light solid; m.p.: 124.3–125.8 °C; ¹H-NMR (DMSO-d₆): δ 1.77 (q, 2H, H-2′, J = 7.0 Hz), 2.22–2.50 (m, 10H, H-3′, H-4′ and H-5′), 2.67 (t, 2H, H-1′, J = 7.4 Hz), 3.33 (s, 2H, H-7′), 3.90 (t, 2H, H-3″, J = 3.8 Hz), 4.26 (t, 2H, H-2″, J = 3.7 Hz), 6.85 (m, 2H, H-6″ and H-8″), 6.95 (t, 1H, H-7″, J = 7.7 Hz), 7.03 (m, 2H, H-5 and H-6), 7.09 (d, 1H, H-2, J = 1.4 Hz), 7.33 (d, 1H, H-4, J = 7.8 Hz), 7.49 (d, 1H, H-7, J = 7.7 Hz), 7.90 (br. s, 1H, H-5″), and 10.79 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.6, 39.2, 40.8, 53.0 (2X), 53.2 (2X), 58.0, 66.6, 111.8, 114.8, 117.2, 118.5, 118.7, 120.1, 121.2, 122.6, 124.3, 126.0, 127.7, 128.5, 136.7, 146.7, and 168.3 ppm. HRMS: (EI) Calculated for C₂₅H₃₀N₄O₂ (M⁺) = 419.5313. Found: 419.4426.

1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7b)

To a solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (237 mg; 0.72 mmol), in dry CH₃CN (50 mL), 7-fluoro-benzoxazine 6b (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added. The chromatographic purification provided 7b (242 mg; 80% yield) as a yellow light solid. m.p.: 124.0–125.3 °C; ¹H-NMR (DMSO-d₆): δ 1.76 (q, 2H, H-2′, J = 7.3 Hz), 2.22–2.48 (m, 10H, H-3′, H-4′, and H-5′), 2.67 (t, 2H, H-1′, J = 7.4 Hz), 3.31 (s, 2H, H-7′), 3.90 (m, 2H, H-3″), 4.29 (m, 2H, H-2″), 6.68–6.78 (m, 2H, H-6″ and H-8″), 6.95 (t, 1H, H-5 or H-6, J = 7.1 Hz), 7.04 (t, 1H, H-6 or H-5, J = 7.0 Hz), 7.09 (d, 1H, H-2, J = 1.8 Hz), 7.32 (d, 1H, H-4, J = 8.0 Hz), 7.49 (d, 1H, H-7, J = 7.7 Hz), 7.92 (br. s, 1H, H-5″), and 10.83 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.6, 53.0 (2X), 53.3 (2X), 58.1, 67, 104.0 (d, ²J_C-F = 26 Hz), 106.9 (d, ^2′J_C-F = 23 Hz), 111.8, 114.8, 118.6 (d, ³J_C-F = 18 Hz), 121.2, 122.6, 123.2, 125.6 (d, ⁴J_C-F = 9 Hz), 127.7, 136.7, 143.2, 144, 147.6, 147.8 (d, ^3′J_C-F = 13 Hz), 159.5 (d, ¹J_C-F = 240 Hz), and 168.2 ppm. HRMS: (EI) Calculated for C₂₅H₂₉FN₄O₂ (M⁺) = 437.5318. Found: 437.5301.

1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7c)

To a solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (237 mg; 0.72 mmol), in dry CH₃CN (50 mL), 6-fluoro-benzoxazine 6c (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added. The crude mixture was column chromatographed to provide pure 7c (269 mg; 89% yield) as a yellow light solid. m.p.: 123.8–125.1 °C; ¹H-NMR (DMSO-d₆): δ 1.83 (q, 2H, H-2′, J = 7.1 Hz), 2.30–2.54 (m, 10H, H-3′, H-4′, and H-5′), 2.73 (t, 2H, H-1′, J = 7.4 Hz), 3.40 (s, 2H, H-7′), 3.98 (t, 2H, H-3″, J = 4.0 Hz), 4.30 (t, 2H, H-2″, J = 3.7 Hz), 6.91–6.97 (m, 2H, H-7″ and H-8″), 7.01 (td, 1H, H-5 or H-6, J_o = 7.3 Hz, J_m = 1.0 Hz), 7.11 (td, 1H, H-6 or H-5, J_o = 7.0 Hz, J_m = 1.1 Hz), 7.15 (d, 1H, H-2, J = 2.1 Hz), 7.38 (d, 1H, H-4, J = 8.0 Hz), 7.55 (d, 1H, H-7, J = 7.7 Hz), 7.95 (br. d, 1H, H-5″, J = 11.7 Hz), and 10.81 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.6, 53.0 (2X), 53.3 (2X), 58.1, 66.1, 110.3 (d, ²J_C-F = 29 Hz), 111.9 (d, ^2′J_C-F = 25 Hz), 114.8, 117.9 (d, ³J_C-F = 9 Hz), 118.5, 118.7, 121.2, 122.6, 126.9 (d, ^3′J_C-F = 12 Hz), 127.7, 128.7, 129.9, 136.7, 138.3, 143 (d, ⁴J_C-F = 2 Hz), 155.5 (d, ¹J_C-F = 239 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C₂₅H₂₉FN₄O₂ (M⁺) = 437.5318. Found: 437.5084.

1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7g)

To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.77 mmol) in CH₃CN (50 mL), 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6a (200 mg; 0.77 mmol) and anhydrous K₂CO₃ (106 mg; 0.77 mmol) were added. The crude mixture was column chromatographed to provide pure 7g (252 mg; 78% yield) as a yellow light solid. m.p.: 168.4–171.3 °C; ¹H-NMR (DMSO-d₆): δ 1.76 (q, 2H, H-2′, J = 6.9 Hz), 2.30–2.51 (m, 10H, H-3′, H-4′ and H-5′), 2.63 (t, 2H, H-1′, J = 7.3 Hz), 3.34 (s, 2H, H-7′), 3.89 (m, 2H, H-3″), 4.26 (t, 2H, H-2″, J = 3.6 Hz), 6.75 (td, 1H, H-6, J_o = 10.4 Hz, J_m = 2.7 Hz), 6.80–6.91 (m, 2H, H-6″, and H-8″), 7.01 (t, 1H, H-7″, J = 7.4 Hz), 7.18 (d, 1H, H-2, J = 1.8 Hz), 7.23 (dd, 1H, H-7, J_o = 10.2 Hz, J_m = 2.3 Hz), 7.30 (dd, 1H, H-4, J_o = 8.9 Hz, J_m = 4.5 Hz), 7.88 (br. s, 1H, H-5″), and 10.89 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.6, 27.2, 52.7 (2X), 53.1 (2X), 57.7, 66.1, 103.4 (d, ²J_C-F = 23 Hz), 104.2, 109.3 (d, ^2′J_C-F = 26 Hz),112.6 (d, ³J_C-F = 9.9 Hz), 115.1 (d, ⁴J_C-F = 4.9 Hz), 117.2, 120.1, 124.3, 124.9, 125.7, 126.6, 127.9 (d, ^3′J_C–F = 9.9 Hz), 133.4, 146.7, 147.9, 157.3 (d, ¹J_C-F = 234 Hz), and 168.2 ppm. HRMS: (EI) Calculated for C₂₅H₂₉FN₄O₂ (M⁺) = 437.5318. Found: 437.5312.

1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7h)

To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.72 mmol) in CH₃CN (50 mL), 1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6b (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added. The crude mixture was column chromatographed to provide pure 7h (205 mg; 65% yield) as a yellow light solid. m.p.: 168.1–169.9 °C; ¹H-NMR (DMSO-d₆): δ 1.75 (q, 2H, H-2′, J = 7.0 Hz), 2.25–2.52 (m, 10H, H-3′, H-4′ and H-5′), 2.64 (t, 2H, H-1′, J = 7.4 Hz), 3.35 (s, 2H, H-7′), 3.92 (t, 2H, H-3″, J = 3.8 Hz), 4.25 (t, 2H, H-2″, J = 3.7 Hz), 6.85–6.93 (m, 3H, H-6, H-6″, and H-8″), 7.19 (d, 1H, H-2, J = 2.1 Hz), 7.24 (dd, 1H, H-7, J_o = 10.2 Hz, J_m = 2.3 Hz), 7.31 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.5 Hz), 7.92 (br. d, 1H, H-5″, J = 9.6 Hz), and 10.88 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.5, 52.9 (2X), 53.2 (2X), 57.8, 66.1, 103.4 (d, ²J_C-F = 23 Hz), 109.3 (d, ^2′J_C-F = 26 Hz), 110.3 (d, ^2″J_C-F = 28 Hz), 112.0 (d, ^2‴J_C-F = 25 Hz), 112.6 (d, ³J_C-F = 9.9 Hz), 115.1 (d, ⁴J_C-F = 4.9 Hz), 117.9 (d, ^3′J_C-F = 9 Hz), 124.9, 126, 126.9 (d, ^3″J_C–F = 9 Hz), 127.9 (d, ^3‴J_C–F = 9.9 Hz), 128.5, 133.3, 143.0 (d, ^4′J_C-F = 2.3 Hz), 155.5 (d, ¹J_C-F = 234 Hz), 157.0 (d, ^1′J_C–F = 231 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C₂₅H₂₈F₂N₄O₂ (M⁺) = 455.5222. Found: 455.5198.

1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7i)

To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.72 mmol) in CH₃CN (50 mL), 1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6c (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added to afford pure 7i (196 mg; 62% yield) as a yellow light solid. m.p.: 166.8–168.1 °C; ¹H-NMR (DMSO-d₆): δ 1.75 (q, 2H, H-2′, J = 7.0 Hz), 2.22–2.55 (m, 10H, H-3′, H-4′ and H-5′), 2.64 (t, 2H, H-1′, J = 7.4 Hz), 3.35 (s, 2H, H-7′), 3.92 (t, 2H, H-3″, J = 3.8 Hz), 4.25 (t, 2H, H-2″, J = 3.7 Hz), 6.83–6.94 (m, 3H, H-6, H-7″ and H-8″), 7.19 (d, 1H, H-2, J = 2.1 Hz), 7.24 (dd, 1H, H-7, J_o = 10.2 Hz, J_m = 2.3 Hz), 7.31 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.5 Hz), 7.92 (br. d, 1H, H-5″, J = 9.6 Hz), and 10.88 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.5, 52.9 (2X), 53.2 (2X), 57.8, 66.1, 103.4 (d, ²J_C-F = 23 Hz), 109.3 (d, ^2′J_C-F = 26 Hz), 110.3 (d, ^2″J_C-F = 28 Hz), 112.0 (d, ^2‴J_C-F = 24.7 Hz), 112.6 (d, ³J_C-F = 9.9 Hz), 115.1 (d, ⁴J_C-F = 4.9 Hz), 117.9 (d, ^3″J_C-F = 8.8 Hz), 124.9, 126.9 (d, ^3′J_C–F = 13 Hz), 126, 127.9 (d, ^3‴J_C–F = 9.9 Hz), 129, 133.3, 143.0 (d, ^4′J_C-F = 2.2 Hz), 155.5 (d, ¹J_C-F = 234 Hz), 157.0 (d, ^1′J_C–F = 231 Hz), and 168.6 ppm. HRMS: (EI) Calculated for C₂₅H₂₈F₂N₄O₂ (M⁺) = 455.5222. Found: 455.5205.

1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7m)

To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (314 mg; 0.77 mmol) in CH₃CN (50 mL), 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6a (200 mg; 0.77 mmol) and anhydrous K₂CO₃ (106 mg; 0.77 mmol) were added. The crude mixture was column chromatographed to provide pure 7m (157 mg; 42% yield) as a yellow light solid. m.p.: 90.5–92.4 °C; ¹H-NMR (DMSO-d₆): δ 1.74 (q, 2H, H-2′, J = 7.1 Hz), 2.25–2.51 (m, 10H, H-3′, H-4′, and H-5′), 2.65 (t, 2H, H-1′, J = 7.1 Hz), 3.34 (s, 2H, H-7′), 3.90 (t, 2H, H-3″, J = 7.1 Hz), 4.27 (t, 2H, H-2″, J = 7.1 Hz), 6.81–6.90 (m, 2H, H-6″, and H-8″), 7.02 (t, 1H, H-7″, J = 7.6 Hz), 7.10–7.20 (m, 2H, H-2, and H-6), 7.30 (d, 1H, H-7, J = 8.5 Hz), 7.68 (d, 1H, H-4, J = 1.6 Hz), 7.85 (br. s, 1H, H-5″), and 11.04 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.6, 52.9 (2X), 53.2 (2X), 57.6, 66.6, 111.2, 113.9, 114.7, 117.2, 120.1, 121.1, 123.6, 124.3, 124.5, 125.7, 126, 126.6, 128.5, 129.6, 135.3, 146.7, and 168.2 ppm. HRMS: (EI) Calculated for C₂₅H₂₉BrN₄O₂ (M⁺) = 498.4374. Found: 498.4362.

1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7n)

To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (294 mg; 0.72 mmol) in CH₃CN (50 mL), 1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6b (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added. The crude mixture was column chromatographed to provide pure 7n (180 mg; 50% yield) as a yellow light solid. m.p.: 96.1–98.1 °C; ¹H-NMR (DMSO-d₆): δ 1.73 (q, 2H, H-2′, J = 6.6 Hz), 2.18–2.51 (m, 10H, H-3′, H-4′and H-5′), 2.64 (t, 2H, H-1′, J = 7.0 Hz), 3.32 (s, 2H, H-7′), 3.90 (m, 2H, H-3″), 4.29 (m, 2H, H-2″), 6.65–6.81 (m, 2H, H-6″ and H-8″), 7.10–7.20 (m, 2H, H-2, and H-6), 7.30 (d, 1H, H-7, J = 8.5 Hz), 7.68 (d, 1H, H-4, J = 1.0 Hz), 7.95 (br. s, 1H, H-5″), and 11.05 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.6, 52.9 (2X), 53.2 (2X), 57.6, 66.6, 110.3 (d, ²J_C-F = 28.5 Hz), 111.2, 112.0 (d, ^2′J_C-F = 25 Hz),113.8, 114.7, 117.9 (d, ³J_C-F = 8.8 Hz), 121.1, 123.6, 124.4, 126.9 (d, ^3′J_C-F = 12 Hz), 129.6, 135.3, 143.0 (d, ⁴J_C-F = 2.3 Hz), 155.5 (d, ¹J_C-F = 233 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C₂₅H₂₈BrFN₄O₂ (M⁺) = 516.4278. Found: 516.4267.

1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7o)

To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (294 mg; 0.72 mmol) in CH₃CN (50 mL), 1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6c (200 mg; 0.72 mmol) and anhydrous K₂CO₃ (100 mg; 0.72 mmol) were added. The crude mixture was column chromatographed to provide pure 7o (251 mg; 70% yield) as a yellow light solid. m.p.: 97.2–98.8 °C; ¹H-NMR (DMSO-d₆): δ 1.74 (q, 2H, H-2′, J = 6.7 Hz), 2.21–2.52 (m, 10H, H-3′, H-4′ and H-5′), 2.65 (t, 2H, H-1′, J = 7.0 Hz), 3.35 (s, 2H, H-7′), 3.92 (m, 2H, H-3″), 4.24 (m, 2H, H-2″), 6.84–6.93 (m, 2H, H-7″ and H8″), 7.10–7.21 (m, 2H, H-2 and H-6), 7.30 (d, 1H, H-7, J = 8.6 Hz), 7.68 (s, 1H, H-4), 7.85 (br. d, 1H, H-5″, J = 7.8 Hz), and 11.04 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.6, 52.9 (2X), 53.2 (2X), 57.7, 66.1, 110.3 (d, ²J_C-F = 28.5 Hz), 111.2, 112.0 (d, ^2′J_C-F = 22.5 Hz), 113.8, 114.7, 117.9 (d, ³J_C-F = 8.8 Hz), 121.1, 123.6, 124.4, 126, 126.9 (d, ^3′J_C-F = 12.1 Hz), 128.5, 129.6, 132.2, 135.3, 138.2, 143.0 (d, ⁴J_C-F = 2.2 Hz), 155.5 (d, ¹J_C-F = 233 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C₂₅H₂₈BrFN₄O₂ (M⁺) = 516.4278. Found: 516.4272.

3.1.4. General procedure for the synthesis of 2,3-Dihydro-benzo[1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamides 7d–f and 7j–l. Method 2

Synthesis of 1-(6-Chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1- piperazinyl} ethanamide. Compound (7d) as a Model

To a stirred solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (224 mg; 0.92 mmol), in dry CH₃CN (50 mL), 2-Chloro-1-(6-chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4e (227 mg; 0.92 mmol), and anhydrous K₂CO₃ (127 mg; 0.92 mmol) were added. The mixture was heated at 80 °C for 24 h. After this time, the resulting mixture was poured into water (100 mL) and extracted with EtOAc (4 × 50 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The organic crude was purified by column chromatography EtOAc/MeOH (6:1) to give pure 7d (290 mg; 71.6% yield) as a yellow light solid. m.p.: 135.3–136.1 °C; ¹H-NMR (DMSO-d₆): δ 1.78 (q, 2H, H-2′, J = 7.0 Hz), 2.20–2.52 (m, 10H, H-3′, H-4′ and H-5′), 2.68 (t, 2H, H-1′, J = 7.3 Hz), 3.32 (s, 2H, H-7′), 3.91 (t, 2H, H-3″, J = 3.7 Hz), 4.27 (t, 2H, H-2″, J = 3.8 Hz), 6.90 (d, 1H, H-8″, J = 8.8 Hz), 6.96 (t, 1H, H-5 or H-6, J = 7.3 Hz), 7.02–7.12 (m, 3H, 2-H, H-6 or H-5 and H-7″), 7.34 (d, 1H, H-4, J = 8.0 Hz), 7.50 (d, 1H, H-7, J = 7.8 Hz), 8.09 (br. s, 1H, H-5″), and 10.75 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.6, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 58.0, 66.4, 111.8, 114.9, 118.5, 118.6, 118.7, 121.2, 122.6, 123.5, 123.6, 125.2, 127.6, 127.7, 136.8, 145.5, and 168.6 ppm. HRMS: (EI) Calculated for C₂₅H₂₉ClN₄O₂ (M⁺) = 453.9864. Found: 453.9856.

1-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7e)

To a solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (241 mg; 0.99 mmol) in CH₃CN (50 mL), 2-Chloro-1-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4d (287 mg; 0.99 mmol) and anhydrous K₂CO₃ (137 mg; 0.99 mmol) were added to afford 7e (298 mg; 62.6% yield) as a yellow light solid. m.p.: 132.7–134.1 °C; ¹H-NMR (DMSO-d₆): δ 1.77 (q, 2H, H-2′, J = 6.7 Hz), 2.27–2.51 (m, 10H, H-3′, H-4′ and H-5′), 2.68 (t, 2H, H-1′, J = 7.3 Hz), 3.32 (s, 2H, H-7′), 3.91 (m, 2H, H-3″), 4.27 (t, 2H, H-2″, J = 3.8 Hz), 6.86 (d, 1H, H-8″, J = 8.8 Hz), 6.96 (td, 1H, H-5 or H-6, J_o = 7.3 Hz, J_m = 1.0 Hz), 7.05 (td, 1H, H-6 or H-5, J_o = 7.3 Hz, J_m = 1.0 Hz), 7.10 (d, 1H, H-2, J = 1.9 Hz), 7.19 (dd, 1H, H-7″, J_o = 8.7 Hz, J_m = 2.1 Hz), 7.33 (d, 1H, H-4, J = 8.0 Hz), 7.50 (d, 1H, H-7, J = 7.8 Hz), 8.20 (br. s, 1H, H-5″), and 10.77 (br.s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.6, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 58.0, 66.5, 111.1, 111.8, 114.8, 118.5, 118.7, 119.1, 121.2, 122.6, 125.4, 126.3, 127.6, 128.1, 136.7, 146.0, and 168.6 ppm. HRMS: (EI) Calculated for C₂₅H₂₉BrN₄O₂ (M⁺) = 498.4373. Found: 498.4364.

1-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7f)

To a solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (282 mg; 1.16 mmol) in CH₃CN (50 mL), 2-Chloro-1-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4f (280 mg; 1.16 mmol) and anhydrous K₂CO₃ (160 mg; 1.16 mmol) were added to provide pure 7f (400 mg; 79% yield) as a yellow light solid. m.p.: 123.6–125.1 °C; ¹H-NMR (DMSO-d₆): δ 1.75 (q, 2H, H-2′, J = 7.0 Hz), 2.23–2.49 (m, 10H, H-3′, H-4′ and H-5′), 2.65 (t, 2H, H-1′, J = 7.4 Hz), 3.31 (s, 2H, H-7′), 3.65 (s, 3H, H-6″), 3.86 (t, 2H, H-3″, J = 3.8 Hz), 4.18 (t, 2H, H-2″, J = 3.8 Hz), 6.63 (dd, 1H, H-7″, J_o = 8.9 Hz, J_m = 2.3 Hz), 6.79 (d, 1H, H8″, J = 8.9 Hz), 6.94 (t, 1H, H-5 or H-6, J = 7.0 Hz), 7.03 (t, 1H, H-6 or H-5, J = 6.9 Hz), 7.08 (d, 1H, H-2, J = 1.9 Hz), 7.31 (d, 1H, H-4, J = 8.0 Hz), 7.48 (d, 1H, H-7, J = 7.8 Hz), 7.61 (br. s, 1H, H-5″), and 10.74 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.8, 27.6, 39.3, 40.5, 53.0 (2X), 53.2 (2X), 55.8, 58.0, 66.1, 109.3, 111.7, 114.8, 117.3, 118.4, 118.7, 121.1, 122.5, 126.8, 127.4, 127.6, 136.7, 140.7, 152.6, and 168.3 ppm. HRMS: (EI) Calculated for C₂₆H₃₂N₄O₃ (M⁺) = 449.5673. Found: 449.5669.

1-(6-Chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7j)

To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (340 mg; 1.3 mmol) in CH₃CN (50 mL), 2-Chloro-1-(6-chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4e (320 mg; 1.3 mmol) and anhydrous K₂CO₃ (180 mg 1.3 mmol) were added to afford pure compound 7j (540 mg; 91% yield) as a yellow light solid. m.p.: 120.5–122.8 °C; ¹H-NMR (DMSO-d₆): δ 1.75 (q, 2H, H-2′, J = 7.1 Hz), 2.21–2.50 (m, 10H, H-3′, H-4′ and H-5′), 2.64 (t, 2H, H-1′, J = 7.4 Hz), 3.33 (s, 2H, H-7′), 3.91 (t, 2H, H-3″,J = 3.7 Hz), 4.27 (t, 2H, H-2″, J = 3.7 Hz), 6.85–6.92 (m, 2H, H-6 and H-8″), 7.07 (dd, 1H, H-7″, J_o = 8.7 Hz, J_m = 2.3 Hz), 7.19 (d, 1H, H-2, J = 1.7 Hz), 7.24 (dd, 1H, H-7, J_o= 10.2 Hz, J_m = 2.3 Hz), 7.31 (dd, 1H, H-4, J_o = 8.9 Hz, J_m = 4.7 Hz), 8.07 (br. s, 1H, H-5″), and 10.88 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.5, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 57.8, 66.4, 103.4 (d, ²J_C-F = 23 Hz), 109.3 (d, ^2′J_C-F = 26.3 Hz), 112.6 (d, ³J_C-F = 9.9 Hz), 114.4, 115.1 (d, ⁴J_C-F = 5.4 Hz), 118.7, 123.5, 124.8, 125.2, 127.5, 127.9 (d, ^3′J_C–F = 9.3 Hz), 133.3, 145.5, 157.0 (d, ¹J_C–F = 231 Hz), and 168.6 ppm. HRMS: (EI) Calculated for C₂₅H₂₈ClFN₄O₂ (M⁺) = 471.9769. Found: 471.9767.

1-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7k)

To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (784 mg; 3 mmol) in CH₃CN (50 mL), 2-Chloro-1-(6-bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4d (870 mg; 3 mmol) and anhydrous K₂CO₃ (180 mg 1.3 mmol) were added to afford pure compound 7k (1130 mg; 75.3% yield) as a yellow light solid. m.p.: 102.8–103.9 °C; ¹H-NMR (DMSO-d₆): δ 1.80 (q, 2H, H-2′, J = 7.0 Hz), 2.26–2.56 (m, 10H, H-3′, H-4′ and H-5′), 2.69 (t, 2H, H-1′, J = 7.3 Hz), 3.38 (s, 2H, H-7′), 3.97 (t, 2H, H-3″,J = 3.4 Hz), 4.33 (t, 2H, H-2″, J = 3.6 Hz), 6.89–6.99 (m, 2H, H-6, and H-8″), 7.13 (dd, 1H, H-7″, J_o = 8.7 Hz, J_m = 2.3 Hz), 7.24 (d, 1H, H-2, J = 1.9 Hz), 7.30 (dd, 1H, H-7, J_o = 10.2 Hz, J_m = 2.3 Hz), 7.37 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.5 Hz), 8.13 (br. s, 1H, H-5″), and 10.93 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.5, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 57.9, 66.4, 103.4 (d, ²J_C-F = 23.1 Hz), 109.3 (d, ^2′J_C-F = 26 Hz), 112.6 (d, ³J_C-F = 9.9 Hz), 115.1 (d, ⁴J_C-F = 4.9 Hz), 118.7, 123.4, 123.5, 124.8, 125.3, 127.6, 127.9 (d, ^3′J_C–F = 9.3 Hz), 133.3, 145.5, 157.0 (d, ¹J_C–F = 231 Hz), and 168.6 ppm. HRMS: (EI) Calculated for C₂₅H₂₈BrFN₄O₂ (M⁺) = 516.4278. Found: 516.4270.

1-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7l)

To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (379 mg; 1.45 mmol) in CH₃CN (50 mL), 2-chloro-1-(6-methoxy-2,3-dihydro-benzo[b][1,4] oxazin-4-yl) ethanamide 4f (350 mg; 1.45 mmol) and anhydrous K₂CO₃ (200 mg; 1.45 mmol) were added to afford pure compound 7l (535 mg; 81.6% yield) as a yellow light solid. m.p.: 94.5–97.3 °C; ¹H-NMR (DMSO-d₆): δ 1.75 (q, 2H, H-2′, J = 7.0 Hz), 2.2–2.50 (m, 10H, H-3′, H-4′ and H-5′), 2.64 (t, 2H, H-1′, J = 7.4 Hz), 3.34 (s, 2H, H-7′), 3.68 (s, 3H, H-6″), 3.89 (t, 2H, H-3″,J = 4.5 Hz), 4.21 (t, 2H, H-2″, J = 3.9 Hz), 6.65 (dd, 1H, H-7″, J_o = 8.9 Hz, J_m = 2.9 Hz), 6.81 (d, 1H, H-8″, J = 8.9 Hz), 6.89 (td, 1H, H-6, J_o = 9.2 Hz, J_m = 2.5 Hz), 7.19 (d,1H, H-2, J = 2.2 Hz), 7.25 (dd, 1H, H-7, J_o = 10.1 Hz, J_m = 2.5 Hz), 7.32 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.6 Hz), 7.62 (br. s, 1H, H-5″), and 10.88 (br. s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.6, 39.3, 40.5, 53.1 (2X), 53.3 (2X), 55.9, 57.9, 66.2, 103.4 (d, ²J_C-F = 23.2 Hz), 109.3 (d, ^2′J_C-F = 26 Hz), 109.3, 112.6 (d, ³J_C-F = 10 Hz), 115.2 (d, ⁴J_C-F = 4.4 Hz), 117.4, 124.9, 126.9, 127.9 (d, ^3′J_C–F = 10 Hz), 128.6, 133.4, 140.7, 152.7, 157.0 (d, ¹J_C–F = 231 Hz), and 168.4 ppm. HRMS: (EI) Calculated for C₂₆H₃₁FN₄O₃ (M⁺) = 467.5577. Found: 467.5567.

3.1.5. General Procedure for the Synthesis of N-(2-morpholin-4-yl-ethyl)-benzamides nitro-fluorinated Derivatives 10a–d

4-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide (10a) as a Model

To a solution of 4-Fluoro-2-nitro-benzoyl chloride (828 mg; 4.07 mmol) in dry THF (50 mL), 2-morpholin-4-yl-ethylamine (0.53 mL; 4.07 mmol) was added. The mixture was stirred at 0 °C under nitrogen atmosphere for 2 h. After this time, saturated solution of NaHCO₃ was added (100 mL) and the mixture was extracted with EtOAc (3 × 60 mL). The organic layers were dried over anhydrous Na₂SO₄ filtered, and concentrated under vacuum conditions to give a crude form, which was purified by column chromatography with EtOAc/MeOH (6:1) as eluent to give 10a (963 mg; 80% yield) as a yellow light solid. m.p.: 149.8–151.5 °C; ¹H-NMR (acetone-d₆): δ 2.32 (t, 4H, H-4′, J = 4.6 Hz), 2.42 (t, 2H, H-3′, J = 6.3 Hz), 3.35 (q, 2H, H-2′, J = 6.3 Hz), 3.46 (t, 4H, H-5′, J = 4.5 Hz), 7.45 (td, 1H, H-5, J_o = 8.2 Hz, J_m = 2.5 Hz), 7.59 (dd, 1H, H-6, J_o = 8.5 Hz, J_m = 5.5 Hz), 7.65 (br. s, 1H, H-1′), and 7.70 (dd, 1H, H-3, J_o = 8.5 Hz, J_m = 2.6 Hz) ppm. ¹³C-NMR (acetone-d₆): δ 36.1, 53 (2X), 56.6, 66 (2X), 111.3 (d, 2J_C-F = 27 Hz), 119.6 (d, 2′J_C-F = 22 Hz), 129.1 (d, 4J_C-F = 3.3 Hz), 130.5 (d, 3J_C-F = 9 Hz), 148.1 (d, 3′J_C-F = 8.2 Hz), 161.7 (d, 1J_C-F = 251 Hz), and 164.2 ppm. HRMS: (EI) Calculated for C₁₃H₁₆FN₃O₄ (M⁺) = 298.1203. Found: 298.1198.

4-Fluoro-N-(2-morpholin-4-yl-ethyl)-3-nitro-benzamide (10b)

4-Fluoro-3-nitro-benzoyl chloride (553 mg; 2.72 mmol), and 2-morpholin-4-yl-ethylamine (0.35 mL; 2.72 mmol), to afford 10b (712 mg; 88% yield) as a yellow light solid. m.p.: 132.5–133.5 °C; ¹H-NMR (acetone-d₆): δ 2.32 (t, 4H, H-4′, J = 4.4 Hz), 2.43 (t, 2H, H-3′, J = 6.6 Hz), 3.37–3.50 (m, 6H, H-2′ and H-5′), 7.48 (m, 1H, H-5), 7.92 (br. s, 1H, H-1′), 8.16 (m, 1H, H-2), and 8.45 (dd, 1H, H-6, J_o = 7.2 Hz, J_m = 2.3 Hz) ppm. ¹³C-NMR (acetone-d₆): δ 36.4, 53.1 (2X), 56.9, 66.1 (2X), 118.1 (d, ²J_C-F = 21.4 Hz), 124.6 (d, ⁴J_C-F = 1.6 Hz), 131.6 (d, ³J_C-F = 3.8 Hz), 134.2 (d, ^2′J_C-F = 9.9 Hz), 150.4 (d, ^3′J_C-F = 3.9 Hz), 156.2 (d, ¹J_C-F = 266 Hz), and 163 ppm. HRMS: (EI) Calculated for C₁₃H₁₆FN₃O₄ (M⁺) = 298.1203, Found: 298.1201.

5-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide (10c)

5-Fluoro-2-nitro-benzoyl chloride (803 mg; 3.94 mmol), and 2-morpholin-4-yl-ethylamine (0.51 mL; 3.94 mmol), to afford 10c (951 mg; 81% yield) as a yellow light solid. m.p.: 103.4–104.5 °C; ¹H-NMR (acetone-d₆): δ 2.33 (t, 4H, H-4′, J = 4.6 Hz), 2.44 (t, 2H, H-3′, J = 6.3 Hz), 3.36 (m, 2H, H-2′), 3.46 (t, 4H, H-5′, J = 4.5 Hz), 7.23–7.37 (m, 2H, H-3, and H-4), 7.67 (br. s, 1H, H-1′), and 8.02 (dd, 1H, H-6, J_o = 9.1 Hz, J_m = 4.8 Hz) ppm. ¹³C-NMR (acetone-d₆): δ 36.1, 53 (2X), 56.5, 66 (2X), 115.6 (d, ²J_C-F = 25.3 Hz), 116.5 (d, ^2′J_C-F = 23.6 Hz), 126.8 (d, ³J_C-F = 9.9 Hz), 136.0 (d, ^3′J_C-F = 8.8 Hz), 143 (d, ⁴J_C-F = 3.3 Hz), 163.9, and 163.9 (d, ¹J_C-F = 256 Hz) ppm. HRMS: (EI) Calculated for C₁₃H₁₆FN₃O₄ (M⁺) = 298.1203. Found: 298.1201.

2-Fluor-N-(2-morpholin-4-yl-ethyl)-5-nitro-benzamide (10d)

2-Fluoro-5-nitro-benzoyl chloride (825 mg; 4.05 mmol), and 2-morpholin-4-yl-ethylamine (0.53 mL; 4.05 mmol), to afford 10d (902 mg; 75% yield) as a yellow light solid. m.p.: 126.4–127.2 °C; ¹H-NMR (acetone-d₆): δ 2.35 (t, 4H, H-4′, J = 4.5 Hz), 2.46 (t, 2H, H-3′, J = 6.3 Hz), 3.43 (q, 2H, H-2′, J = 6.5 Hz), 3.49 (t, 4H, H-5′, J = 4.5 Hz), 7.43 (m, 1H, H-3), 7.66 (br. s, 1H, H-1′), 8.30 (m, 1H, H-4), and 8.56 (dd, 1H, H-6, J_o = 6.3 Hz, J_m = 3 Hz) ppm. ¹³C-NMR (acetone-d₆): δ 36.1, 52.9 (2X), 56.2, 66.1 (2X), 117.4 (d, ²J_C-F = 26.9 Hz), 123.6 (d, ^2′J_C-F = 16.5 Hz), 126.3 (d, ⁴J_C-F = 4.9 Hz), 127.5 (d, ³J_C-F = 11 Hz), 144(d, ^3′J_C-F = 9 Hz), 160.4, and 162.8 (d, ¹J_C-F = 259 Hz) ppm. HRMS: (EI) Calculated for C₁₃H₁₆FN₃O₄ (M⁺) = 298.1203. Found: 298.1201.

3.1.6. General Procedure for the Synthesis of N-(2-morpholin-4-yl-ethyl)-benzamides amino-fluorinated Derivatives 11a–d

2-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide (11a) as a Model

To a mixture containing water-acetic acid-ethanol (1:1:1), 4-Fluoro-N-(2-morpholin-4-yl-ethyl)-2- nitro-benzamide 10a (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) were added. The resulting mixture was heated and stirred for 3 h at 70 °C. After this time, the mixture was filtered to remove excess metallic iron, transferred to a flask containing a mixture of EtOAc/H₂O (400 mL, 1:1), and neutralized with NaHCO₃ (10 gr). The aqueous phase was extracted with EtOAc (50 mL × 3). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under vacuum to give a crude, which was purified by column chromatography with EtOAc/MeOH (6:1) to give 11a (891 mg; 98% yield) as a yellow light solid. m.p.: 120.3–121.7 °C; ¹H-NMR (CDCl₃): δ 2.46 (t, 4H, H-4′, J = 4.4 Hz), 2.55 (t, 2H, H-3′, J = 6.1 Hz), 3.46 (q, 2H, H-2′, J = 5.6 Hz), 3.69 (t, 4H, H-5′, J = 4.7 Hz), 5.72 (br. s, 2H, H-2), 6.28–6.35 (m, 2H, H-3 and H-5), 6.57 (br. s, 1H, H-1′), and 7.25 (dd, 1H, H-6, J_o = 9.3 Hz, J_m = 3.2 Hz) ppm. ¹³C-NMR (CDCl₃): δ 36.7, 53.3 (2X), 56.7, 66.9 (2X), 102.3 (d, ²J_C-F = 24.2 Hz), 103.9 (d, ^2′J_C-F = 23 Hz), 112.4 (d, ⁴J_C-F = 2.2 Hz), 129.3 (d, ³J_C-F = 11 Hz), 151.1 (d, ^3′J_C-F = 11.7 Hz), 165.3 (d, ¹J_C-F = 249 Hz), and 168.6 ppm. HRMS: (EI) Calculated for C₁₃H₁₈FN₃O₂ (M⁺) = 268.1461. Found: 268.1454.

3-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide (11b)

4-Fluoro-N-(2-morpholin-4-yl-ethyl)-3-nitro-benzamide 10b (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) to afford 11b (726 mg; 81% yield) as a yellow light solid. m.p.: 147.4–149.1 °C; ¹H-NMR (DMSO-d₆): δ 2.40–2.51 (m, 4H, H-4′), 2.57 (br. s, 2H, H-3′), 3.40 (q, 2H, H-2′, J = 6.6 Hz), 3.63 (t, 4H, H-5′, J = 4.3 Hz), 5.39 (br. s, 2H, H-3), 6.99–7.15 (m, 2H, H-2 and H-4), 7.31 (dd, 1H, H-6, J_o = 8.9 Hz, J_m = 1.6 Hz), and 6.57 (br. t, 1H, H-1′, J = 5.2 Hz), ppm. ¹³C-NMR (DMSO-d₆): δ 36.9, 53.7 (2X), 57.8, 66.6 (2X), 115.0 (d, ²J_C-F = 30 Hz), 115.1 (d, ³J_C-F = 3.3 Hz), 116 (d, ^3′J_C-F = 6 Hz), 131.7 (d, ⁴J_C-F = 2.8 Hz), 136.7 (d, ^2′J_C-F = 13.2 Hz), 152.5 (d, ¹J_C-F = 242 Hz), and 168.3 ppm. HRMS: (EI) Calculated for C₁₃H₁₈FN₃O₂ (M⁺) = 268.1461. Found: 268.1458.

2-Amino-N-(2-morpholin-4-yl-ethyl)-5-fluoro-benzamide (11c)

5-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide 10c (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) to afford 11c (697 mg; 78% yield) as a yellow light solid. m.p.: 104.8–105.6 °C; ¹H-NMR (CDCl₃): δ 2.47 (t, 4H, H-4′, J = 4.4 Hz), 2.55 (t, 2H, H-3′, J = 6.1 Hz), 3.47 (q, 2H, H-2′, J = 5.6 Hz), 3.69 (t, 4H, H-5′, J = 4.6 Hz), 5.28 (br. s, 2H, H-2), 6.55–6.63 (br. m, 2H, H-3, and H-1′), 6.93 (td, 1H, H-6, J_o = 8.6 Hz, J_m = 2.9 Hz), and 7.0 (dd, 1H, H-4, J_o = 9.1 Hz, J_m = 2.7 Hz) ppm. ¹³C-NMR (CDCl₃): δ 35.9, 53.3 (2X), 56.8, 67 (2X), 113.1 (d, ²J_C-F = 23 Hz), 116.6 (d, ³J_C-F = 5.1 Hz), 118.4 (d, ^3′J_C-F = 7.3 Hz), 119.5 (d, ^2′J_C-F = 23 Hz), 144.8 (d, ⁴J_C-F = 1.5 Hz), and 155.5 (d, ¹J_C-F = 236 Hz), 168.3 ppm. HRMS: (EI) Calculated for C₁₃H₁₈FN₃O₂ (M⁺) = 268.1461. Found: 268.1459.

5-Amino-N-(2-morpholin-4-yl-ethyl)-2-fluoro-benzamide (11d)

2-Fluoro-N-(2-morpholin-4-yl-ethyl)-5-nitro-benzamide 10d (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) to afford 11d (798 mg; 89% yield) as a yellow light solid. m.p.: 106.8–107.9 °C; ¹H-NMR (CDCl₃): δ 2.37 (t, 4H, H-4′, J = 4.4 Hz), 2.45 (t, 2H, H-3′, J = 6.1 Hz), 3.24–3.50 (br. m, 4H, H-5 H-2′), 3.59 (t, 4H, H-5′, J = 4.7 Hz), 6.59 (m, 1H, H-6), 6.78 (m, 1H, H-3), 7.23 (dd, 1H, H-4, J_o = 6.4 Hz, J_m = 3.2 Hz), and 7.28 (br. s, 1H, H-1′) ppm. ¹³C-NMR (CDCl₃): δ 36.3, 53.2 (2X), 56.5, 67 (2X), 116.6 (d, ²J_C-F = 26.4 Hz), 117.0 (d, ⁴J_C-F = 1.5 Hz), 119.1 (d, ³J_C-F = 8.8 Hz), 121.3 (d, ^2′J_C-F = 13.2 Hz), 143.1 (d, ^3′J_C-F = 2.2 Hz), 154.2 (d, ¹J_C-F = 238 Hz), and 163.4 ppm. HRMS: (EI) Calculated for C₁₃H₁₈FN₃O₂ (M⁺) = 268.1461. Found: 268.1457.

3.1.7. General Procedure for the Synthesis of (2-Chloro-acetylamino)-N-(2-morpholin-4-yl-ethyl) fluorinated Benzamides Derivatives 12a–d

2-(2-Chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12a) as a Model

To a solution of 2-amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide 11a (400 mg; 1.5 mmol) in dry THF (60 mL), 2-chloro-acetylchloride (0.12 mL; 1.5 mmol) was added. The mixture was stirred at 0 °C under nitrogen atmosphere for 2 h. After this time, a saturated solution of NaHCO₃ (100 mL) was added. The mixture was extracted with EtOAc (3 X 100 mL). The organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum conditions to give a crude, which was purified by column chromatography EtOAc/MeOH (6:1), to give 12a (479 mg; 93% yield) as a white solid. m.p.: 124.8–126.3 °C; ¹H-NMR (CDCl₃): δ 2.48 (t, 4H, H-4′, J = 4.4 Hz), 2.58 (t, 2H, H-3′, J = 5.9 Hz), 3.51 (q, 2H, H-2′, J = 5.4 Hz), 3.69 (t, 4H, H-5′, J = 4.6 Hz), 4.13 (s, 2H, H-1″), 6.81 (td, 1H, H-5, J_o = 7.7 Hz, J_m = 2.7 Hz), 6.88 (br. s, 1H, H-1′), 7.46 (dd, 1H, H-5, J_o = 8.8 Hz, J_m = 6.1 Hz), 8.42 (dd, 1H, H-3, J_o = 11.6 Hz, J_m = 2.7 Hz), and 12.24 (br. s, 1H, H-2) ppm. ¹³C-NMR (CDCl₃): δ 36, 43.2, 53.3 (2X), 56.5, 66.9 (2X), 108.7 (d, ²J_C-F = 28 Hz), 110.6 (d, ^2′J_C-F = 22 Hz), 116.8 (d, ⁴J_C-F = 3.7 Hz), 128.4 (d, ³J_C-F = 10.3 Hz), 141 (d, ^3′J_C-F = 12.5 Hz), 164.7 (d, ¹J_C-F = 252 Hz), 165.5, and 167.8 ppm. HRMS: (EI) Calculated for C₁₅H₁₉ClFN₃O₃ (M⁺) = 344.1177. Found: 344.1173.

3-(2-Chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12b)

3-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide 11b (400 mg; 1.5 mmol) and 2-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12b (482 mg; 94% yield) as a white solid. m.p.: 129.1–130.8 °C; ¹H-NMR (CDCl₃): δ 2.48 (t, 4H, H-4′, J = 4.4 Hz), 2.57 (t, 2H, H-3′, J = 6.1 Hz), 3.50 (q, 2H, H-2′, J = 5.6 Hz), 3.71 (t, 4H, H-5′, J = 4.7 Hz), 4.20 (s, 2H, H-1″), 6.89 (br. s, 1H, H-1′), 7.14–7.19 (m, 1H, H-5), 7.64–7.68 (m, 1H, H-2), 8.57 (br. s, 1H, H-3), and 8.67 (dd, 1H, H-6, J_o = 7.3 Hz, J_m = 2 Hz) ppm. ¹³C-NMR (CDCl₃): δ 36.2, 42.9, 53.3 (2X), 56.6, 66.9 (2X), 115.4 (d, ²J_C-F = 19.8 Hz), 119.5 (d, ⁴J_C-F = 1.5 Hz), 125.2 (d, ³J_C-F = 8.8 Hz), 125.4 (d, ^2′J_C-F = 11 Hz), 131.4 (d, ^3′J_C-F = 2.9 Hz), 154.3 (d, ¹J_C-F = 250 Hz), 164.1, and 166 ppm. HRMS: (EI) Calculated for C₁₅H₁₉ClFN₃O₃ (M⁺) = 344.1177. Found: 344.1176.

2-(2-Chloro-acetylamino)-5-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12c)

2-Amino-N-(2-morpholin-4-yl-ethyl)-5-fluoro-benzamide 11c (400 mg; 1.5 mmol) and 2-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12c (496 mg, 97% yield) as a white solid. m.p.: 98.5–99.8 °C; ¹H-NMR (CDCl₃): δ 2.55 (t, 4H, H-4′, J = 4.4 Hz), 2.65 (t, 2H, H-3′, J = 6.1 Hz), 3.57 (c, 2H, H-2′, J = 5.6 Hz), 3.77 (t, 4H, H-5′, J = 4.7 Hz), 4.20 (s, 2H, H-1″), 6.93 (br. s, 1H, H-1′), 7.19–7.27 (m, 2H, H-4 and H-6), 8.61 (dd, 1H, H-3, J_o = 8.9 Hz, J_m = 5.1 Hz), and 11.8 (br. s, 1H, H-2) ppm. ¹³C-NMR (CDCl₃): δ 36.1, 43.1, 53.3 (2X), 56.5, 66.9 (2X), 113.3 (d, ²J_C-F = 24 Hz), 119.2 (d, ⁴J_C-F = 22 Hz), 122.7 (d, ³J_C-F = 5.9 Hz), 123.6 (d, ^2′J_C-F = 7.3 Hz), 134.8 (d, ^3′J_C-F = 2.9 Hz), 158.2 (d, ¹J_C-F = 245 Hz), 165.1, and 167.3 ppm. HRMS: (EI) Calculated for C₁₅H₁₉ClFN₃O₃ (M⁺) = 344.1177. Found: 344.1164.

5-(2-Chloro-acetylamino)-2-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12d)

5-Amino-N-(2-morpholin-4-yl-ethyl)-2-fluoro-benzamide 11d (400 mg; 1.5 mmol) and 2-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12d (483 mg; 94% yield) as a white solid. m.p.: 144.1–145.1 °C; ¹H-NMR (CDCl₃): δ 2.48 (t, 4H, H-4′, J = 4.2 Hz), 2.57 (t, 2H, H-3′, J = 5.9 Hz), 3.54 (q, 2H, H-2′, J = 5.4 Hz), 3.69 (t, 4H, H-5′, J = 4.4 Hz), 4.15 (s, 2H, H-1″), 7.05–7.13 (m, 1H, H-3), 7.47 (br. s, 1H, H-1′), 7.9 (dd, 1H, H-4, J_o = 6.5 Hz, J_m = 2.7 Hz), 8.03–8.09 (m, 1H, H-6), and 8.70 (br. s, 1H, H-5) ppm. ¹³C-NMR (CDCl₃): δ 36.4, 42.9, 53.2 (2X), 56.3, 67 (2X), 116.9 (d, ²J_C-F = 26.4 Hz), 121.5 (d, ^2′J_C-F = 13.2 Hz), 123.1 (d, ⁴J_C-F = 2.2 Hz), 125.2 (d, ³J_C-F = 8.8 Hz), 133.9 (d, ^3′J_C-F = 2.9 Hz), 157.4 (d, ¹J_C-F = 246 Hz), 162.6, and 164.4 ppm. HRMS: (EI) Calculated for C₁₅H₁₉ClFN₃O₃ (M⁺) = 344.1177. Found: 344.1173.

3.1.8. General Procedure for the Synthesis of (2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl)-fluorinated Benzamides Derivatives 13a–l

4-Fluoro-2-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13a) as a Model

To a stirred solution of 5-fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), in dry CH₃CN (50 mL), 2-(2-chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12a (199 mg; 0.58 mmol) and anhydrous K₂CO₃ (80 mg; 0.58 mmol) were added. The mixture was heated at 80 °C for 24 h. After this time, the resulting mixture was poured into water (100 mL), extracted with EtOAc (4 × 50 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The organic crude was purified by column chromatography EtOAc/MeOH (6:1) to give 13a (298 mg; 62.6%) as a yellow light solid. m.p.: 91.3–93.1 °C; ¹H-NMR (DMSO-d₆): δ 1.78 (q, 2H, H-2′, J = 7.4 Hz), 2.26–2.56 (m, 16H, H-3′, H-4′, H-5′, H-9″ and H-1‴), 2.66 (t, 2H, H-1′, J = 7.3 Hz), 3.13 (s, 2H, H-6′), 3.45–3.62 (m, 6H, H-8′, and H-2‴), 6.90 (td, 1H, H-6, J_o = 9.2 Hz, J_m = 2.5 Hz), 7.01 (td, 1H, H-5″, J_o = 8.4 Hz, J_m = 2.7 Hz), 7.20 (d, 1H, H-2, J = 2.1 Hz), 7.25 (dd, 1H, H-7, J_o = 10.2 Hz, J_m = 2.5 Hz), 7.32 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.7 Hz), 7.68–7.76 (m, 1H, H-3″), 8.40 (dd, 1H, H-6″, J_o = 12.3 Hz, J_m = 2.6 Hz), 8.62 (t, 1H, H-7″, J = 5.5 Hz), 10.87 (s, 1H, H-1), and 12.24 (br. s, 1H, H-7′) ppm. ¹³C-NMR (DMSO-d₆): δ 22.2, 27.0, 36.4, 52.4 (2X), 53.0 (2X), 53.2 (2X), 57.1, 57.5, 61.8, 66.1 (2X), 102.7 (d, ²J_C-F = 23.1 Hz), 106.5 (d, ^2′J_C-F = 28.2 Hz), 108.8 (d, ^2″J_C-F = 26 Hz), 109.1 (d, ^2‴J_C-F = 22.3 Hz), 112.1 (d, ³J_C-F = 9.5 Hz) 114.7 (d, ⁴J_C-F = 4.8 Hz), 118.2 (d, ^4′J_C-F = 2.9 Hz), 124.3, 127.4 (d, ^3′J_C-F = 9.5 Hz), 130.2 (d, ^3″J_C–F = 9.9 Hz), 132.9, 140.2 (d, ^3‴J_C-F = 12.1 Hz), 156.5 (d, ¹J_C–F = 231 Hz), 163.3 (d, ^1′J_C-F = 246 Hz), 166.8, and 169.8 ppm. HRMS: (EI) Calculated for C₃₀H₃₈F₂N₆O₃ (M⁺) = 569.3051. Found: 569.3050.

4-Fluoro-3-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13b)

5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 3-(2-chloro-acetylamino)- 4-fluor-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13b (150 mg; 47% yield) as a yellow light solid. m.p.: 92.2–93.9 °C; ¹H-NMR (DMSO-d₆): δ 1.78 (q, 2H, H-2′, J = 7.4 Hz), 2.27–2.56 (m, 16H, H-3′, H-4′, H-5′, H-9″ and H-1‴), 2.66 (t, 2H, H-1′, J = 7.3 Hz), 3.19 (s, 2H, H-6′), 3.33–3.36 (m, 2H, H-8′), 3.57 (t, 4H, H-2‴, J = 4.4 Hz), 6.89 (td, 1H, H-6, J_o = 9.2 Hz, J_m = 2.5 Hz), 7.20 (d, 1H, H-2, J = 1.9 Hz), 7.26 (dd, 1H, H-7, J_o = 10.1 Hz, J_m = 2.4 Hz), 7.29–7.42 (m, 2H, H-4 and H-5″), 7.59–7.67 (m, 1H, H-2″), 8.40–8.50 (m, 2H, H-6″, and H-7″), 9.68 (br. s, 1H, H-7′), and 10.87 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.5, 37.1, 53.3 (2X), 53.4 (2X), 53.7 (2X), 57.7, 57.9, 61.6, 66.7 (2X), 103.4 (d, ²J_C-F = 23.5 Hz), 109.3 (d, ^2″J_C-F = 26.4 Hz), 112.6 (d, ^3′J_C-F = 9.5 Hz), 115.2 (d, ⁴J_C-F = 4.4 Hz), 115.6 (d, ^2′J_C-F = 19.8 Hz), 123 (d, ^4′J_C-F = 6.6 Hz), 124.5 (d, ³J_C-F = 8.1 Hz), 124.9, 126.2 (d, ^2‴J_C-F = 11.7 Hz), 127.9 (d, ^3′J_C–F = 9.5 Hz), 131.6 (d, ³J_C-F = 2.9 Hz), 133.4, 155.3 (d, ¹J_C-F = 248 Hz), 157.0 (d, ^1′J_C–F = 230.4 Hz). 165.6, and 169.1 ppm. HRMS: (EI) Calculated for C₃₀H₃₈F₂N₆O₃ (M⁺) = 569.3051. Found: 569.3046.

2-Fluoro-5-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13c)

5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 5-(2-Chloro-acetylamino)- 2-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13c (178 mg; 55% yield) as a yellow light solid. m.p.: 91.8–93.2 °C; ¹H-NMR (DMSO-d₆): δ 1.76 (q, 2H, H-2′, J = 7.4 Hz), 2.32 (t, 2H, H-3′, J = 6.9 Hz), 2.36–2.56 (m, 14H, H-4′, H-5′, H-9″ and H-1‴), 2.65 (t, 2H, H-1′, J = 7.3 Hz), 3.11 (s, 2H, H-6′), 3.36–3.42 (m, 2H, H-8″), 3.57 (t, 4H, H-2‴, J = 4.5 Hz), 6.89 (td, 1H, H-6, J_o = 8.2 Hz, J_m = 2.6 Hz), 7.17–7.28 (m, 3H, H-2, H-7 and H-3″), 7.31 (dd, 1H, H-4, J_o = 8.8 Hz, J_m = 4.6 Hz), 7.73–7.81 (m, 1H, H-6″), 7.92 (dd, 1H, H-4″, J_o = 6.5 Hz, J_m = 2.8 Hz), 8.13–8.24 (m, 1H, H-7″), 9.90 (br. s, 1H, H-7′), and 10.89 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.7, 27.6, 37, 53.1 (2X), 53.4 (2X), 53.7 (2X), 57.4, 57.9, 62.3, 66.7 (2X), 103.4 (d, ²J_C-F = 22.7 Hz), 109.3 (d, ^2′J_C-F = 26.5 Hz), 112.6 (d, ^3″J_C-F = 9.9 Hz), 115.2 (d, ⁴J_C-F = 4.4 Hz), 116.7 (d, ^2″J_C-F = 23.8 Hz), 121.33 (d, ^4′J_C-F = 2.2 Hz), 123.6 (d, ^2‴J_C-F = 8.3 Hz), 124.2 (d, ³J_C-F = 9.5 Hz), 124.9, 127.9 (d, ^3‴J_C–F = 9.4 Hz), 133.6, 135.5 (d, ^3′J_C-F = 3.3 Hz), 155.4 (d, ¹J_C–F = 245 Hz), 157 (d, ^1′J_C-F = 231 Hz), 163.7, and 168.9 ppm. HRMS: (EI) Calculated for C₃₀H₃₈F₂N₆O₃ (M⁺) = 569.3051. Found: 569.3047.

5-Fluoro-2-(2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13d)

5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 2-(2-chloro- acetylamino)-5-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13d (188 mg; 59% yield) as a yellow light solid. m.p.: 94.2–95.4 °C; ¹H-NMR (DMSO-d₆): δ 1.79 (q, 2H, H-2′, J = 7.2 Hz), 2.32–2.51 (m, 16H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.66 (t, 2H, H-1′, J = 7.3 Hz), 3.10 (s, 2H, H-6′), 3.50–3.60 (m, 6H, H-8″, and H-2‴), 6.89 (td, 1H, H-6, J_o = 9.2 Hz, J_m = 2.5 Hz), 7.20 (d, 1H, H-2, J = 2.0 Hz), 7.25 (dd, 1H, H-7, J_o = 10.1 Hz, J_m = 2.5 Hz), 7.29–7.40 (m, 2H, H-4 and H-4″), 7.49 (dd, 1H, H-3″, J_o = 9.5 Hz, J_m = 3.0 Hz), 8.53 (dd, 1H, H-6″, J_o = 9.2 Hz, J_m = 5.4 Hz), 8.69 (t, 1H, H-7″, J = 5.4 Hz), 10.89 (s, 1H, H-1), and 11.62 (br. s, 1H, H-7′) ppm. ¹³C-NMR (DMSO-d₆): δ 22.2, 26.9, 36.4, 52.4 (2X), 52.9 (2X), 53.2 (2X), 57, 57.4, 61.7, 66.1 (2X), 102.9 (d, ²J_C-F = 22.3 Hz), 108.8 (d, ^2′J_C-F = 26.3 Hz), 112.1 (d, ³J_C-F = 9.9 Hz), 114.5 (d, ^2″J_C-F = 23.8 Hz), 114.6 (d, ⁴J_C-F = 5 Hz), 118 (d, ^2‴J_C-F = 22 Hz), 122.1 (d, ^3′J_C-F = 7 Hz), 123.8 (d, ^3″J_C-F = 6.2 Hz), 124.3, 127.4 (d, ^3‴J_C–F = 9.5 Hz), 132.9, 134.4 (d, ^4′J_C-F = 2.2 Hz), 152.4 (d, ¹J_C–F = 231 Hz), 156.5 (d, ^1′J_C-F = 223 Hz), 166.3, and 169 ppm. HRMS: (EI) Calculated for C₃₀H₃₈F₂N₆O₃ (M⁺) = 569.3051. Found: 569.3048.

4-Fluoro-2-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13e)

5-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 2-(2-chloro-acetylamino)-4- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12a (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13e (236 mg; 66% yield) as a yellow light solid. m.p.: 92.1 -93.8 °C; ¹H-NMR (DMSO-d₆): δ 1.78 (q, 2H, H-2′, J = 7.4 Hz), 2.35 (t, 2H, H-3′, J = 6.9 Hz), 2.38–2.52 (m, 14H, H-4′, H-5′, H-9″ and H-1‴), 2.67 (t, 2H, H-1′, J = 7.3 Hz), 3.13 (s, 2H, H-6′), 3.40–3.42 (m, 2H, H-8″), 3.54 (t, 4H, H-2‴, J = 4.5 Hz), 7.0 (td, 1H, H-5″, J_o = 8.3 Hz, J_m = 2.8 Hz), 7.12–7.20 (m, 2H, H-2, and H-6), 7.31 (d, 1H, H-7, J = 8.6 Hz), 7.62 (d, 1H, H-4, J = 1.7 Hz), 7.77 (dd, 1H, H-3″, J_o = 8.8 Hz, J_m = 6.6 Hz), 8.39 (dd, 1H, H-6″, J_o = 12.3 Hz, J_m = 2.7 Hz), 8.70 (t, 1H, H-7″, J = 5.4 Hz), 11.1 (s, 1H, H-1), and 12.1 (br. s, 1H, H-7′) ppm. ¹³C-NMR (DMSO-d₆): δ 22.8, 27.4, 36.9, 52.6 (2X), 52.7 (2X), 53.7 (2X), 56.2, 57.6, 62.2, 66.6 (2X), 107.1 (d, ²J_C-F = 26.5 Hz), 109.7 (d, ^2′J_C-F = 22.7 Hz), 111.2, 113.3, 118.6 (d, ⁴J_C-F = 4.9 Hz), 118.7, 121.6, 123.7, 124.4, 129.6, 130.8 (d, ³J_C-F = 9.9 Hz), 135.4, 140.7 (d, ^3′J_C-F = 12.2 Hz), 163.8 (d, ¹J_C-F = 246 Hz), 167.3, and 170.4 ppm. HRMS: (EI) Calculated for C₃₀H₃₈BrFN₆O₃ (M⁺) = 629.2251. Found: 629.2246.

4-Fluoro-3-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13f)

5-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 3-(2-chloro-acetylamino)- 4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13f (193 mg; 54% yield) as a yellow light solid. m.p.: 85.2–86.8 °C; ¹H-NMR (DMSO-d₆): δ 1.78 (q, 2H, H-2′, J = 6.5 Hz), 2.30–2.55 (m, 16H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.68 (t, 2H, H-1′, J = 7.3 Hz), 3.20 (s, 2H, H-6′), 3.40–3.44 (m, 2H, H-8″), 3.57 (t, 4H, H-2‴, J = 4.4 Hz), 7.13–7.21 (m, 2H, H-2, and H-6), 7.31 (d, 1H, H-7, J = 10.1 Hz), 7.34–7.42 (m, 1H, H-5″), 7.59–7.67 (m, 1H, H-2″), 7.62 (d, 1H, H-4, J = 1.6 Hz), 8.37–8.52 (m, 2H, H-6″ and H-7″), 9.69 (br. s, 1H, H-7′), and 11.0 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.2, 26.9, 36.4, 52.4 (2X), 52.9 (2X), 53.2 (2X), 57, 57.4, 61.7, 66.1 (2X), 111.3, 113.8, 114.7, 115.6 (d, ²J_C-F = 21 Hz), 121.1, 123.1, 123.28 (d, ⁴J_C-F = 2.9 Hz), 123.7, 124.5 (d, ³J_C-F = 9.5 Hz), 126.2 (d, ^2′J_C-F = 11 Hz), 129.7, 131.63 (d, ^3′J_C-F = 3.7 Hz), 135.7, 155.3 (d, ¹J_C-F = 248 Hz), 165.6, and 169 ppm. HRMS: (EI) Calculated for C₃₀H₃₈BrFN₆O₃ (M⁺) = 629.2251. Found: 629.2250.

2-Fluoro-5-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13g)

5-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 5-(2-chloro-acetylamino)-2- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13g (249 mg; 70% yield) as a yellow light solid. m.p.: 73.2–74.5 °C; ¹H-NMR (DMSO-d₆): δ 1.79 (q, 2H, H-2′, J = 7.2 Hz), 2.32–2.51 (m, 16 H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.66 (t, 2H, H-1′, J = 7.3 Hz), 3.10 (s, 2H, H-6′), 3.50–3.60 (m, 6H, H-8″, and H-2‴), 6.89 (td, 1H, H-6, J_o = 9.2 Hz, J_m = 2.5 Hz), 7.20 (d, 1H, H-2, J = 2.0 Hz), 7.25 (dd, 1H, H-7, J_o = 10.1 Hz, J_m = 2.5 Hz), 7.29–7.40 (m, 2H, H-4, and H6″), 7.49 (dd, 1H, H-4″, J_o = 9.5 Hz, J_m = 3.0 Hz), 8.53 (dd, 1H, H-3″, J_o = 9.2 Hz, J_m = 5.4 Hz), 8.69 (t, 1H, H-7″, J = 5.4 Hz), 10.89 (s, 1H, H-1), and 11.62 (br. s, 1H, H-7′) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.5, 36.9, 52.9 (2X), 53 (2X), 53.6 (2X), 574, 57.6, 62.1, 66.7 (2X), 111.3, 113.8, 114.7, 116.7 (d, ²J_C-F = 23.5 Hz), 121.1, 121.4 (d, ³J_C-F = 3.7 Hz), 123.6 (d, ^3′J_C-F = 7.3 Hz), 123.7, 124.2 (d, ^2′J_C-F = 15.4 Hz), 124.5, 129.6, 135.3, 135.4 (d, ⁴J_C-F = 2.2 Hz), 155.4 (d, ¹J_C-F = 245 Hz), 163.7, and 168.9 ppm. HRMS: (EI) Calculated for C₃₀H₃₈BrFN₆O₃ (M⁺) = 629.2251. Found: 629.2248.

5-Fluoro-2-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13h)

5-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 2-(2-chloro-acetylamino)-5- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (199 mg; 0.58 mmol), and anhydrous K₂CO₃ (80 mg; 0.58 mmol), to afford 13h (303 mg; 85% yield) as a yellow light solid. m.p.: 73.8–74.7 °C; ¹H-NMR (DMSO-d₆): δ 1.71–1.84 (m, 2H, H-2′), 2.25–2.56 (m, 16H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.67 (t, 2H, H-1′, J = 7.2 Hz), 3.14 (s, 2H, H-6′), 3.40–3.43 (m, 2H, H-8″), 3.57 (t, 4H, H-2‴, J = 4.5 Hz), 7.13–7.27 (m, 3H, H-2, and H-6 and H-3″), 7.31 (d, 1H, H-7, J = 8.5 Hz), 7.69 (d, 1H, H-4, J = 1.7 Hz), 7.73–7.81 (m, 1H, H-6″), 7.92 (dd, 1H, H-4″, J_o = 6.4 Hz, J_m = 2.7 Hz), 8.15–8.22 (m, 1H, H-7″), 9.9 (br. s, 1H, H-7′), and 11 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.5, 36.9, 52.8 (2X), 53.3 (2X), 53.7 (2X), 57.5, 57.7, 62.2, 66.6 (2X), 111.3, 113.8, 114.7, 115.1 (d, ²J_C-F = 24.2 Hz), 118.5 (d, ^2′J_C-F = 21.3 Hz), 121.1, 122.6 (d, ³J_C-F = 7.3 Hz), 123.7, 124.3 (d, ^3′J_C-F = 5.9 Hz), 124.4, 129.6, 134.9 (d, ⁴J_C-F = 2.2 Hz), 135.4, 157.3 (d, ¹J_C-F = 241 Hz), 166.9, and 169.5 ppm. HRMS: (EI) Calculated for C₃₀H₃₈BrFN₆O₃ (M⁺) = 629.2251. Found: 629.2249.

4-Fluoro-2-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13i)

3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 2-(2-chloro-acetylamino)-4-fluoro- N-(2-morpholin-4-yl-ethyl)-benzamide 12a (213 mg; 0.62 mmol), and anhydrous K₂CO₃ (86 mg; 0.62 mmol), to afford 13i (249 mg; 75% yield) as a yellow light solid. m.p.: 80.5–81.8 °C; ¹H-NMR (DMSO-d₆): δ 1.86 (m, 2H, H-2′), 2.25–2.49 (m, 12H, H-3′, H-4′, H-9″and H-1‴), 2.58 (m, 4H, H-5′), 2.7 (t, 2H, H-1′, J = 7.4 Hz), 3.15 (s, 2H, H-6′), 3.35–3.40 (m, 2H, H-8″), 3.54 (t, 4H, H-2‴, J = 4.3 Hz), 6.93–7.09 (m, 3H, H-5 and H-6 and H-5″), 7.12 (d, 1H, H-2, J = 1 Hz), 7.33 (d, 1H, H-7, J = 8 Hz), 7.51 (d, 1H, H-4, J = 7.8 Hz), 7.76 (dd, 1H, H-3″, J_o = 8.6 Hz, J_m = 6.7 Hz), 8.39 (dd, 1H, H-6″, J_o = 12.3 Hz, J_m = 2.6 Hz), 8.68 (t, 1H, H-7″, J = 5 Hz), 10.8 (s, 1H, H-1), and 12.1 (br. s, 1H, H-7′) ppm. ¹³C-NMR (DMSO-d₆): δ 22.5, 27.7, 36.9, 52.9 (2X), 53.5 (2X), 53.7 (2X), 55.4, 57.6, 62.3, 66.6 (2X), 107 (d, ²J_C-F = 28 Hz), 109.7 (d, ^2′J_C-F = 22 Hz), 111.8, 114.5, 118.6 (d, ⁴J_C-F = 2.9 Hz), 121.3, 122.6, 127.6, 130.8 (d, ³J_C-F = 10.9 Hz), 134, 136.8, 140.8 (d, ^3′J_C-F = 12.5 Hz), 141.4, 163.8 (d, ¹J_C-F = 245 Hz), 167.3, and 170.3 ppm. HRMS: (EI) Calculated for C₃₀H₃₉FN₆O₃ (M⁺) = 551.3145. Found: 551.3139.

4-Fluoro-3-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13j)

3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 3-(2-chloro-acetylamino)-4- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (213 mg; 0.62 mmol), and anhydrous K₂CO₃ (86 mg; 0.62 mmol), to afford 13j (268 mg; 81% yield) as a yellow light solid. m.p.: 70.2–71.9 °C; ¹H-NMR (DMSO-d₆): δ 1.81 (q, 2H, H-2′, J = 7.5 Hz), 2.21–2.53 (m, 16H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.7 (t, 2H, H-1′, J = 7.4 Hz), 3.19 (s, 2H, H-6′), 3.38–3.40 (m, 2H, H-8″), 3.57 (t, 4H, H-2‴, J = 4.5 Hz), 6.96 (td, 1H, H-5 or H-6, J_o = 7.5 Hz, J_m = 1 Hz), 7.06 (td, 1H, H-6 or H-5, J_o = 7.4 Hz, J_m = 1 Hz), 7.11 (d, 1H, H-2, J = 2.1 Hz), 7.32–7.40 (m, 2H, H-7 and H-5″), 7.51 (d, 1H, H-4, J = 7.7 Hz), 7.61–7.66 (m, 1H, H-2″), 8.40–8.52 (m, 2H, H-6″ and H-7″), 9.69 (br. s, 1H, H-7′), and 10.8 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.4, 27.1, 36.6, 52.8 (2X), 52.9 (2X), 53.2 (2X), 57.2, 57.5, 61.1, 66.2 (2X), 111.3, 114.3, 115.1 (d, ²J_C-F = 20.1 Hz), 118.1 (d, ³J_C-F = 10.6 Hz), 120.7, 122.1, 122.4, 123.0, 124.0 (d, ^3′J_C-F = 8.1 Hz), 125.7 (d, ^2′J_C-F = 11.8 Hz), 127.2, 131.1 (d, ⁴J_C-F = 3.3 Hz), 136.3, 154.7 (d, ¹J_C-F = 249 Hz), 165.1, and 168.5 ppm. HRMS: (EI) Calculated for C₃₀H₃₉FN₆O₃ (M⁺) = 551.3145. Found: 551.3140.

2-Fluoro-5-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13k)

3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 5-(2-chloro-acetylamino)-2- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (213 mg; 0.62 mmol), and anhydrous K₂CO₃ (86 mg; 0.62 mmol), to afford 13k (200 mg; 60.7% yield) as a yellow light solid. m.p.: 69.7–71.5 °C; ¹H-NMR (DMSO-d₆): δ 1.81 (q, 2H, H-2′, J = 6.8 Hz), 2.25–2.54 (m, 16H, H-3′, H-4′, H-5′, H-9″, and H-1‴), 2.7 (t, 2H, H-1′, J = 7.2 Hz), 3.13 (s, 2H, H-6′), 3.39 (q, 2H, H-8″, J = 6.4 Hz), 3.58 (t, 4H, H-2‴, J = 4.2 Hz), 6.96 (t, 1H, H-5 or H-6, J = 7.2 Hz), 7.06 (t, 1H, H-6 or H-5, J = 6.9 Hz), 7.11 (d, 1H, H-2, J = 1.2 Hz), 7.24 (t, 1H, H-3″, J = 9.8 Hz), 7.34 (d, 1H, H-4, J = 8.0 Hz), 7.51 (d, 1H, H-7, J = 7.7 Hz), 7.76–7.81 (m, 1H, H-6″), 7.94 (dd, 1H, H-4″, J_o = 6.3 Hz, J_m = 2.6 Hz), 8.15–8.22 (m, 1H, H-7″), 9.90 (br. s, 1H, H-7′), and 10.8 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.5, 37.0, 53.0 (2X), 53.2 (2X), 53.7 (2X), 57.4, 57.9, 62.2, 66.7 (2X), 111.8, 114.8, 116.7 (d, ²J_C-F = 24.2 Hz), 118.5, 118.7, 121.2, 121.4 (d, ⁴J_C-F = 2.2 Hz), 112.6, 123.6 (d, ³J_C-F = 8.1 Hz), 124.1 (d, ^2′J_C-F = 16.1 Hz), 127.7, 135.4 (d, ³J_C-F = 2.9 Hz), 136.8, 155.4 (d, ¹J_C-F = 245 Hz), 163.7, and 168.9 ppm. HRMS: (EI) Calculated for C₃₀H₃₉FN₆O₃ (M⁺) = 551.3145. Found: 551.3142.

5-Fluoro-2-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl) Benzamide (13l)

3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (200 mg; 0.82 mmol), 2-(2-chloro-acetylamino)-5- fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (281 mg; 0.82 mmol), and anhydrous K₂CO₃ (113 mg; 0.82 mmol), to afford 13l (263 mg; 60% yield) as a yellow light solid. m.p.: 68.1–69.6 °C; ¹H-NMR (DMSO-d₆): δ 1.80 (q, 2H, H-2′, J = 7.6 Hz), 2.30–2.52 (m, 16H, H-3′, H-4′, H-5′, H-9″ and H-1‴), 2.69 (t, 2H, H-1′, J = 7.4 Hz), 3.18 (s, 2H, H-6′), 3.70–3.41 (m, 2H, H-8″), 3.56 (t, 4H, H-2‴, J = 4.5 Hz), 6.96 (td, 1H, H-5 or H-6, J_o = 7.8 Hz, J_m = 1.0 Hz), 7.05 (td, 1H, H-6 or H-5, J_o = 7 Hz, J_m= 1.1 Hz), 7.11 (d, 1H, H-2, J = 2.1 Hz), 7.33 (t, 1H, H-4″, J = 8.4 Hz), 7.39 (d, 1H, H-4, J = 8.7 Hz), 7.50 (d, 1H, H-7, J = 7.8 Hz), 7.59–7.66 (m, 1H, H-3″), 8.40–8.48 (m, 2H, H-6″ and H-7″), 9.68 (br. s, 1H, H-7′), and 10.75 (s, 1H, H-1) ppm. ¹³C-NMR (DMSO-d₆): δ 22.9, 27.7, 37.1, 53.3 (2X), 53.4 (2X), 53.8 (2X), 57.8, 58.1, 61.6, 66.7 (2X), 111.7, 113.4, 114.9, 115.6 (d, ²J_C-F = 19.4 Hz), 118.6 (d, ^2′J_C-F = 17.7 Hz), 121.2, 122.6, 123.0, 124.5 (d, ³J_C-F = 8.3 Hz),126.2 (d, ^3′J_C-F = 11.6 Hz), 127.7, 131.63 (d, ⁴J_C-F = 2.8 Hz), 136.7, 155.3 (d, ¹J_C-F = 249 Hz), 165.5, and 169.1 ppm. HRMS: (EI) Calculated for C₃₀H₃₉FN₆O₃ (M⁺) = 551.3145. Found: 551.3141.

3.2. Biological Assay

3.2.1. Reagents

[³H]Paroxetine (20.8 Ci/mmol; Code NET869), [³H]-Methylspiperone (specific activity 64.1 Ci/mmol; NET856), membrane from clonal cell line HEK-293 that overexpresses SERT (Code: RBHSTM400UA), and membrane from CHO-K1 clonal cell line that overexpresses D₂ receptor (Code: RBHD2CM400UA) were purchased from Perkin-Elmer (Boston, MA, USA). Fluoxetine and haloperidol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were of analytical grade.

3.2.2. SERT Binding

To determine the binding of all compounds at SERT, competitive binding assays were performed according to previously reported procedures with some modifications [36]. Briefly, assays were carried out in a total volume of 0.5 mL containing 9 µg protein of membrane from a clonal cell line HEK-293 that overexpresses SERT, 50 mM Tris buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 nM [³H]-paroxetine (specific activity 20.8 Ci/mmol, PerkinElmer), and the compounds to be tested at different concentrations (10⁻⁹–10⁻⁴ M). After 1 h at 27 °C, incubations were stopped by rapid filtration through Whatman GF/C filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 mL of ice-cold buffer, dried, and put in Eppendorf tubes with scintillation liquid. Radioactivity was counted by a liquid scintillation counter (MicroBeta 2450 microplate counter, PerkinElmer). Control curve was performed with fluoxetine in the same experimental conditions. Non-specific binding was determined with 10 µM fluoxetine.

3.2.3. D₂ Receptor Binding

To determine the binding of all compounds at D₂ receptor, competitive binding assays were performed according to provider indications with some modifications. Briefly, assays were carried out in a total volume of 0.5 mL containing 3 µg protein of membrane from a CHO-K1 clonal cell line that overexpresses D₂ receptor, 50 mM Tris buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 0.5 nM [³H]-methylspiperone (specific activity 64.1 Ci/mmol, PerkinElmer), and the compounds to be tested at different concentrations (10⁻⁹–10⁻⁴ M). After 2 h at 27 °C, incubations were stopped by rapid filtration through Whatman GF/C filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 mL of ice-cold wash buffer (50 mM Tris buffer, pH 7.4, 154 mM NaCl), dried, and put in Eppendorf tubes with scintillation liquid. Radioactivity was counted as described before. Control curve was performed with haloperidol in the same conditions. Non-specific binding was determined with 10 µM haloperidol.

Analysis of data: All curves were fitted using the sigmoidal dose–response inhibition curve (variable slope) equation built into GraphPad PRISM 5.01 (GraphPad Software Inc., San Diego, CA, USA). The analysis gives the IC₅₀ value (i.e., the drug concentration inhibiting specific binding by 50%) to calculate Ki (affinity constant) by the Cheng–Prussof equation (Ki = IC₅₀/(1 + ([radioligand]/Kd (radioligand))). The Kd values used correspond to 0.13 nM to [³H] paroxetine on SERT [50], and 0.1 nM to [³H]-methylspiperone on D₂ (provided by the manufacturer). The IC₅₀ and Ki values correspond to the results of three independent experiments, each in triplicate. All data are expressed as the mean ± SEM.

3.2.4. MAO-A Inhibition

All experimental procedures were approved by the Ethics Committee of the University of Santiago de Chile and the Science Council (FONDECYT) of Chile and followed internationally accepted guidelines (NIH Guide for the Care and Use of Laboratory Animals).

The effects of the compounds on rat MAO-A activity were studied following a previously reported methodology [50,51], using a crude rat brain mitochondrial suspension as a source of enzyme. Serotonin (100 µM) was used as the selective substrate for MAO-A. This compound and its metabolite were detected by HPLC with electrochemical detection. As an exploratory evaluation, the percentage of MAO-A inhibition in the presence of 100 µM of the different compounds was determined, with the idea of evaluating in detail those compounds showing an inhibitory activity in the range of 70–100%.

3.2.5. Molecular Docking

Molecular docking studies for the two families of compounds were performed on two different protein targets (SERT and D₂ receptor). All dockings were carried out at pH 7.4 in the crystal structures of human SERT (hSERT PDB: 5I73) [47] and human D₂ receptor [hD₂ PDB: 6CM4) [49]. All compounds were modelled using the Spartan’14 Software (Wavefunction, Inc. Irvine, CA) and geometry optimization calculations were carried out using the software package at the Hartree-Fock level using the 6-31G* basis set. Docking studies were performed using AutoDockv4.2 [52] software suite with Autodock Tools ADT 1.5.6 [52,53] following the standard docking procedure for rigid proteins. Grid maps were calculated using the autogrid option with a grid volume of 70 × 70 × 70 points with a grid spacing of 0.375 Å and centered on the coordinates x, y, z: 33.3 184.5; 0.565 −9.397; and 37.019 28.136 for the SERT and D₂ receptor respectively. Docking simulations were performed with a Lamarckian genetic algorithm (LGA) and binding energies were estimated according to the internal scoring function implemented by the program; 250 independent runs per ligand were carried out with an initial population of 300 individuals. Default settings were used for all other parameters. The lowest free-energy resulting complexes were selected and further analyzed using the Visual Molecular Dynamic (VMD) visualization program [54]. Validation of the docking protocol was performed using the co-crystallized ligands (S)-citalopram and risperidone for SERT and D₂, respectively.

3.2.6. QSAR Methods

CoMFA and CoMSIA studies were performed with Sybyl X-1.2 software [55] installed in a Windows 10 environment on a PC with an Intel Core i7 CPU. The geometric optimization, field calculation, and charges calculation were performed as previously reported [56] (Figure S1 and Table S3 in Supplementary Material) [57]. The internal validation of the models was done by calculating the cross-validation coefficient q² [58]. The models with the highest value of q² were selected and then subjected to external validation [59,60,61] (Table S2). In all cases, the best models passed the validation limits [59] (Table S2). The regression graphs of each model and the tables of experimental versus calculated values are in the Supplementary Material (Table S3, Figure S2).

4. Conclusions

According to these results, the design of hybrid or bifunctional compounds, i.e., molecules that incorporate two pharmacophores known to act at different receptors into a single chemical entity, is an attractive approach for the development of agents having a targeted polypharmacological profile [19,62,63]. In the present work, we attempted to combine SERT effects previously demonstrated for indolylalkylpiperazine derivatives, functionalizing the parent scaffold with structural fragments of drugs with known activity upon D₂ receptor or MAO-A. Unexpectedly, the synthesized compounds did not show, in most cases, a multitarget profile, since they exhibited a high affinity for SERT while showing almost no effect at D₂ receptor or MAO-A. This indicates that this strategy, although plausible, requires a very fine design of the fragments to be connected and how these are going to be linked. Beyond these considerations, our results highlight the remarkable stability of the indolylpropylpiperazine skeleton as SERT ligand, which exhibits a high affinity by this target, apparently regardless of the type of the associated moiety [34,35,36]. We think that this represents an important feature for the design of polypharmacological molecules, in which an effect upon SERT is pursued. Docking and QSAR results allowed us to rationalize the high SERT affinity observed for compounds in both studied families. Thus, the presence of a halogen at the C-5 position of the indole ring and fluorine atoms at the benzoxazine (Series I) or acetanilide (Series II) moieties probably induces electronic deprotection of the corresponding aromatic rings, favoring stronger π–π interactions of these frameworks with donor aromatic residues at the binding site.

Interestingly, one of the compounds (7n) showed a promissory multitarget profile, being the only derivative showing a relatively high and comparable affinity for SERT and D₂ receptor (Ki = 84.4 and 307 nM, respectively). Even though at this time it is difficult to determine the molecular aspects underlying this pharmacological promiscuity, it is clear that for polypharmacological drugs, a similar affinity for different receptors is the most relevant characteristic, and therefore 7n stands as a very attractive lead for further optimization.