Microwave-Assisted Synthesis of Trazodone and Its Derivatives as New 5-HT1A Ligands: Binding and Docking Studies

Trazodone, a well-known antidepressant drug widely used throughout the world, works as a 5-hydroxytryptamine (5-HT2) and α1-adrenergic receptor antagonist and a serotonin reuptake inhibitor. Our research aimed to develop a new method for the synthesis of trazodone and its derivatives. In the known methods of the synthesis of trazodone and its derivatives, organic and toxic solvents are used, and the synthesis time varies from several to several dozen hours. Our research shows that trazodone and its derivatives can be successfully obtained in the presence of potassium carbonate as a reaction medium in the microwave field in a few minutes. As a result of the research work, 17 derivatives of trazodone were obtained, including compounds that exhibit the characteristics of 5-HT1A receptor ligands. Molecular modeling studies were performed to understand the differences in the activity toward 5-HT1A and 5-HT2A receptors between ligand 10a (2-(6-(4-(3-chlorophenyl)piperazin-1-yl)hexyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one) (5-HT1A Ki = 16 nM) and trazodone. The docking results indicate the lack of the binding of ligand 10a to 5-HT2AR, which is consistent with the in vitro studies. On the other hand, the docking results for the 5-HT1A receptor indicate two possible binding modes. Crystallographic studies support the hypothesis of an extended conformation.

In summary, the methods of the synthesis of trazodone, which are known so far, require solvents such as acetonitrile, toluene, dioxane, or isopropyl alcohol, and the synthesis time of the product varies from several to several dozen hours.
As already mentioned, trazodone is a potent serotonin 5-HT2A and α1-adrenergic receptor antagonist.
Although trazodone is a well-known antidepressant, its pharmacological activity is not fully understood, and it is thought to have more than one mechanism of action. There is ample evidence that suggests that the antidepressant activity of trazodone involves serotonin type 2 (5-HT2) receptor antagonism and the inhibition of serotonin transporter, which results in agonistic effects against 5-HT1A. As a result, 5-HT1A, 5-HT2, and other receptors (5-HT6, 5-HT7, and D2) play an important role in these diseases of the central nervous system [6][7][8][9][10][11][12][13][14][15]. In addition, 5-HT1A receptor ligands are of great interest due to the fact that new therapeutic targets were identified, i.e., prostate cancer treatment, and gastrointestinal and cardiopulmonary disorders [16].
We recently reported the synthesis of a number of hexylarylpiperazine derivatives, which showed high affinity for 5-HT1A receptor [17,18]. As a continuation of this study and in order to test whether the extension of the trazodone linker will change the activity profile, we present here the design, synthesis, and biological evaluation of new derivatives of trazodone as potential 5-HT1A ligands. In the research work, we also attempted to adapt our method of synthesis under microwave radiation [5,19], in the synthesis of trazodone itself, as well as of its substrates and its derivatives.
In summary, the methods of the synthesis of trazodone, which are known so far, require solvents such as acetonitrile, toluene, dioxane, or isopropyl alcohol, and the synthesis time of the product varies from several to several dozen hours.
Although trazodone is a well-known antidepressant, its pharmacological activity is not fully understood, and it is thought to have more than one mechanism of action. There is ample evidence that suggests that the antidepressant activity of trazodone involves serotonin type 2 (5-HT 2 ) receptor antagonism and the inhibition of serotonin transporter, which results in agonistic effects against 5-HT 1A . As a result, 5-HT 1A , 5-HT 2 , and other receptors (5-HT 6 , 5-HT 7 , and D 2 ) play an important role in these diseases of the central nervous system [6][7][8][9][10][11][12][13][14][15]. In addition, 5-HT 1A receptor ligands are of great interest due to the fact that new therapeutic targets were identified, i.e., prostate cancer treatment, and gastrointestinal and cardiopulmonary disorders [16].
We recently reported the synthesis of a number of hexylarylpiperazine derivatives, which showed high affinity for 5-HT 1A receptor [17,18]. As a continuation of this study and in order to test whether the extension of the trazodone linker will change the activity profile, we present here the design, synthesis, and biological evaluation of new derivatives of trazodone as potential 5-HT 1A ligands. In the research work, we also attempted to adapt our method of synthesis under microwave radiation [5,19], in the synthesis of trazodone itself, as well as of its substrates and its derivatives.

Synthesis of Trazodone
Our research shows that trazodone can be successfully obtained in the reaction of 2-(3-halopropyl) [1,2,4]triazolo [4,3-a]pyridin-3(2H)-one (3a/b) and 1-(3-chlorophenyl) piperazine hydrochloride (4), carried out in the presence of potassium carbonate as a reaction medium, a PTC (phase transfer catalyst) in the microwave radiation field (Scheme 1, Method I). The same process conditions can be used successfully to synthesize trazodone with the reaction of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) with chloropropyl-2-chloroarylpiperazine (5) (Scheme 1, Method II). In addition, these conditions can be also used in a "one-pot" reaction, without isolating intermediates (Scheme 1, Method III). The research started with the synthesis of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) using two methods (Scheme 2). The first one concerns a two-step process involving the reaction of 2halopyridine (6a/b) with hydrazine in the first stage (yield 14%) in dimethylformamide (DMF) for 120 s. Bearing in mind the low efficiency, the reaction was also carried out under conventional conditions by heating the substrates in ethanol for 25 h [3], which resulted in a 50% yield. In the case of the synthesis of 7 from 6a/b, the higher yield was obtained by classical synthesis. In the next step, the obtained compound 7 was reacted with equimolar amount of urea (yield 49%) or with molar excess (yield 75%) in a solvent-free condition for 50 s. The obtained results, especially in the second stage, are very interesting because the analogous reaction under conventional conditions described in the literature takes 2 h and allows the synthesis of compound 1 with a lower yield (57%) [20].
The second method for the synthesis of 1 in a one-step reaction involves the use of semicarbazide in 2-ethoxyethanol (yield 43-48%). All reactions were carried out under microwave radiation, which is a new way of obtaining 1,2,4-triazolo[4,3-a] pyridin-3(2H)-one (1) and which is yet to be described in the literature.  The research started with the synthesis of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) using two methods (Scheme 2). The first one concerns a two-step process involving the reaction of 2-halopyridine (6a/b) with hydrazine in the first stage (yield 14%) in dimethylformamide (DMF) for 120 s. Bearing in mind the low efficiency, the reaction was also carried out under conventional conditions by heating the substrates in ethanol for 25 h [3], which resulted in a 50% yield. In the case of the synthesis of 7 from 6a/b, the higher yield was obtained by classical synthesis. In the next step, the obtained compound 7 was reacted with equimolar amount of urea (yield 49%) or with molar excess (yield 75%) in a solvent-free condition for 50 s. The obtained results, especially in the second stage, are very interesting because the analogous reaction under conventional conditions described in the literature takes 2 h and allows the synthesis of compound 1 with a lower yield (57%) [20].
The second method for the synthesis of 1 in a one-step reaction involves the use of semicarbazide in 2-ethoxyethanol (yield 43-48%). All reactions were carried out under microwave radiation, which is a new way of obtaining 1,2,4-triazolo[4,3-a] pyridin-3(2H)-one (1) and which is yet to be described in the literature. The research started with the synthesis of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) using two methods (Scheme 2). The first one concerns a two-step process involving the reaction of 2halopyridine (6a/b) with hydrazine in the first stage (yield 14%) in dimethylformamide (DMF) for 120 s. Bearing in mind the low efficiency, the reaction was also carried out under conventional conditions by heating the substrates in ethanol for 25 h [3], which resulted in a 50% yield. In the case of the synthesis of 7 from 6a/b, the higher yield was obtained by classical synthesis. In the next step, the obtained compound 7 was reacted with equimolar amount of urea (yield 49%) or with molar excess (yield 75%) in a solvent-free condition for 50 s. The obtained results, especially in the second stage, are very interesting because the analogous reaction under conventional conditions described in the literature takes 2 h and allows the synthesis of compound 1 with a lower yield (57%) [20].
The second method for the synthesis of 1 in a one-step reaction involves the use of semicarbazide in 2-ethoxyethanol (yield 43-48%). All reactions were carried out under microwave radiation, which is a new way of obtaining 1,2,4-triazolo[4,3-a] pyridin-3(2H)-one (1) and which is yet to be described in the literature.   The research started with the synthesis of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) using two methods (Scheme 2). The first one concerns a two-step process involving the reaction of 2halopyridine (6a/b) with hydrazine in the first stage (yield 14%) in dimethylformamide (DMF) for 120 s. Bearing in mind the low efficiency, the reaction was also carried out under conventional conditions by heating the substrates in ethanol for 25 h [3], which resulted in a 50% yield. In the case of the synthesis of 7 from 6a/b, the higher yield was obtained by classical synthesis. In the next step, the obtained compound 7 was reacted with equimolar amount of urea (yield 49%) or with molar excess (yield 75%) in a solvent-free condition for 50 s. The obtained results, especially in the second stage, are very interesting because the analogous reaction under conventional conditions described in the literature takes 2 h and allows the synthesis of compound 1 with a lower yield (57%) [20].
The second method for the synthesis of 1 in a one-step reaction involves the use of semicarbazide in 2-ethoxyethanol (yield 43-48%). All reactions were carried out under microwave radiation, which is a new way of obtaining 1,2,4-triazolo[4,3-a] pyridin-3(2H)-one (1) and which is yet to be described in the literature.  The reactions were carried out under microwave radiation in the presence of K 2 CO 3 and PTC catalysts (TBAB). Table 1 contains the results of the research including the impact of the solvent (acetonitrile (ACN) and DMF) on the yield and time of obtaining 3a/b. The studies showed that, when the reaction is carried out with DMF or ACN under the same conditions, the obtained efficiency is almost four times better with ACN ( Table 1, entries 1 and 2); therefore, further experiments were carried out with this solvent only. Surprisingly, a subsequent study showed that, when the amount of the solvent was decreased, or even completely eliminated, this did not cause significant differences in the performance (Table 1, entries 2, 3, 4, 7, and 8). The replacement of halogen in the substrate 2a on 2b caused a slight 17% drop in yield (Table 1, entries 4 and 7). In the case of the reaction carried out in a Magnum II reactor in a closed polytetrafluoroethylene (PTFE) tube, similar efficiency was obtained, but it was necessary to extend the time of the process ( Table 1, entries 4 and 5).
In the next stage of the synthesis of trazodone with method I, the  The reactions were carried out under microwave radiation in the presence of K2CO3 and PTC catalysts (TBAB). Table 1 contains the results of the research including the impact of the solvent (acetonitrile (ACN) and DMF) on the yield and time of obtaining 3a/b. The studies showed that, when the reaction is carried out with DMF or ACN under the same conditions, the obtained efficiency is almost four times better with ACN (Table 1, entries 1 and 2); therefore, further experiments were carried out with this solvent only. Surprisingly, a subsequent study showed that, when the amount of the solvent was decreased, or even completely eliminated, this did not cause significant differences in the performance (Table 1, (Table 1, entries 4 and 7). In the case of the reaction carried out in a Magnum II reactor in a closed polytetrafluoroethylene (PTFE) tube, similar efficiency was obtained, but it was necessary to extend the time of the process ( Table 1, entries 4 and 5).
In the next stage of the synthesis of trazodone with method I, the obtained 2-(3halopropyl) [1,2,4] The synthesis of trazodone with the method I was carried out with potassium carbonate and PTC (TBAB-tetrabutylammonium bromide, TEAC-tetraethylammonium chloride, and DABCO-1,4-diazabicyclo[2.2.2]octane). The reactions were carried out under microwave radiation. During the experiment, we observed that the addition of a small amount (<10% weight) of DMF or ACN was beneficial for the duration of the process, but the reaction also took place under completely solventfree conditions (Table 2, entries 5, 6, and 11). The obtained results for the synthesis of trazodone with method I showed that the reaction occurred in less than 5 min, while, in the presence of DMF or ACN, it occurred within 1-2 min. The highest yield was observed in the reaction with 2-(3-bromopropyl)-1,2,4-triazolo[4,3-a]pyridin-3-(2H)-one (3b) using 10% by weight DMF and TBAB as a PTC catalyst, which allowed obtaining a product with 98% efficiency in 1 min (Table 2, entry 2). When the TBAB was exchanged for TEAC or DABCO under the same conditions, the reaction time was longer by 2 The synthesis of trazodone with the method I was carried out with potassium carbonate and PTC (TBAB-tetrabutylammonium bromide, TEAC-tetraethylammonium chloride, and DABCO-1,4-diazabicyclo[2.2.2]octane). The reactions were carried out under microwave radiation. During the experiment, we observed that the addition of a small amount (<10% weight) of DMF or ACN was beneficial for the duration of the process, but the reaction also took place under completely solvent-free conditions ( Table 2, entries 5, 6, and 11). The obtained results for the synthesis of trazodone with method I showed that the reaction occurred in less than 5 min, while, in the presence of DMF or ACN, it occurred within 1-2 min. The highest yield was observed in the reaction with 2-(3-bromopropyl)-1,2,4-triazolo[4,3-a]pyridin-3-(2H)-one (3b) using 10% by weight DMF and TBAB as a PTC catalyst, which allowed obtaining a product with 98% efficiency in 1 min (Table 2, entry 2). When the TBAB was exchanged for TEAC or DABCO under the same conditions, the reaction time was longer by 2 min (Table 2, entries 3 and 4). The exchange of DMF for ACN caused a 13% decrease in yield ( Table 2, entries 1 and 7). Table 2. Synthesis of trazodone with method I-10 mmol of 2-(3-chloropropyl)-1,2,4-triazolo[4,3a]pyridin-3-(2H)-one (3a) /10 mmol of 2-(3-bromopropyl)-1,2,4-triazolo[4,3-a]pyridin-3-(2H)-one (3b), 10 mmol of 1-(3-chlorophenyl) piperazine hydrochloride (4) In the case of the use of 2-(3-chloropropyl)-1,2,4-triazolo[4,3-a]pyridin-3-(2H)-one (3a), the amount of ACN in the range of 10-40% did not significantly affect the efficiency ( Table 2, entries 8-10). For these reactions, higher yields were observed when ACN was used rather than DMF or H 2 O. The effect of pressure on the course of the reaction and the product yield were also evaluated by conducting the process in a closed PTFE tube in a Magnum II reactor. For pressures of 1 and 5 bar, similar results were obtained, while the increase of the pressure to 10 bar resulted in a decreased product efficiency by about 20% (Table 2, entries 14-16).

Method III-One-Pot Synthesis
Trazodone was also obtained in a "one-pot" variant. The process was carried under microwave radiation by firstly heating 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) with 1-bromo-3-chloropropane (2a) for 50 s, followed by the addition of 1-(3-chlorophenyl)piperazine (4) for another 90 s. The expected product was obtained with a yield of 31%. The synthesis was also performed in the CEM Discover SP reactor (100 W), carrying out the process for 30 s in the first stage and for 60 s in the second, which allowed obtaining trazodone with a yield of 71%.
To summarize the synthesis of trazodone under microwave radiation, it can certainly be assumed that this method is applicable at every stage of the synthesis. This method, in comparison to conventional methods, increases the yield of the obtained products, shortens the time of the Scheme 6. Synthesis of trazodone with method II under MW radiation.
The highest efficiency (92%) in the synthesis of trazodone with method II was obtained in the reaction for 80 s using about 15 wt.% ACN (Table 4, entry 4). Table 4.

Method III-One-Pot Synthesis
Trazodone was also obtained in a "one-pot" variant. The process was carried under microwave radiation by firstly heating 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (1) with 1-bromo-3-chloropropane (2a) for 50 s, followed by the addition of 1-(3-chlorophenyl)piperazine (4) for another 90 s. The expected product was obtained with a yield of 31%. The synthesis was also performed in the CEM Discover SP reactor (100 W), carrying out the process for 30 s in the first stage and for 60 s in the second, which allowed obtaining trazodone with a yield of 71%.
To summarize the synthesis of trazodone under microwave radiation, it can certainly be assumed that this method is applicable at every stage of the synthesis. This method, in comparison to conventional methods, increases the yield of the obtained products, shortens the time of the synthesis, excludes or reduces the amount of solvents used in the process, and reduces the amount of energy.
Bearing in mind that the highest yields of trazodone were obtained with method I, as well as the fact that this process can be quite easily scaled-up, we decided to synthesize trazodone derivatives according to this method.

Synthesis of Trazodone Derivatives
The synthesis of trazodone with method I also works very well in the synthesis of its derivatives (Scheme 7, Table 5) (10a-r). The reactions were carried out under similar conditions as the previous ones, i.e., potassium carbonate, TBAB, and a small amount of ACN. All of the syntheses were carried out in the SAMSUNG device (Table 6). Additionally, for the selected ligands, the synthesis in the CEM Discover SP reactor was also performed, giving slightly higher yields (10e, yield = 58%; 10o, yield = 61%; 10r yield = 66%). The obtained products were converted into hydrochlorides.
The derivatives of trazodone (10a-k) with a modified aryl substituent were obtained by the condensation reaction of 2-(3-chloropropyl)-1,2,4-triazolo[4,3-a]pyridin-3-(2H)-one (4a) with appropriate arylpiperazines 7. As already mentioned in the introduction, and bearing in mind the high activity with 5-HT 1A receptors in ligands with the hexyl linker that we previously synthesized [17][18][19]22], hexyl derivatives of trazodone were also obtained (10e-k) (Scheme 7).                 The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). * Percent purity of the purified product was calculated on the peak area integration during HPLC analysis.
The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D2, 5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The progress of the reaction was monitored with thin-layer chromatography (TLC) and the purity of the resulting ligands was assessed with UPLC-MS. The structures were confirmed based on the analysis of data obtained from 1 H NMR, 13 C NMR, and infrared (IR).

Biological Evaluation of Trazodone Derivatives
The ligands obtained in the synthesis (10a-r) were tested in vitro for binding to D 2 , 5-HT 1A , 5-HT 2A , 5-HT 6 , and 5-HT 7 receptors on the basis of the screening protocol described previously [17] ( Table 6). The in vitro results collected in Table 6 show that replacement of the 3-chloroarylpiperazine system in trazodone, as well as chain elongation, results in a decreased activity relative to the 5-HT 2A receptor. Interestingly, extending the chain by three carbon atoms, while maintaining the 3-chloroarylpiperazine system, resulted in a change in the pharmacological profile and binding to the 5-HT 1A receptor (10e). The majority of the synthesized hexyl ligands ( Table 6, entries 5-18) provided a high affinity and selectivity for the 5-HT 1A receptor.
Interestingly, adding chlorine to this structure, at position 3 to position 4 (10h) resulted in a fourfold increase of the activity toward 5-HT 2A and 5-HT 6 . When there was only a substituent in the 4-position (10g), the activity was similar to that in the case of 10e (Table 6). Among ligands with chlorine substitution, the compound with the substitution at position 2 (10f) had the least interesting activity profile. However, when chlorine was replaced with fluorine at this position, the binding and selectivity to 5-HT 1A increased significantly (10i). Among ligands with a hexyl chain and the arylmethoxy group linked to the piperazine moiety, ligands with substitution at positions 2 (10j) and 3 (10) had a much higher binding to 5-HT 1A compared to their 4-substituted analog (10l). Interestingly, if the methoxy group was replaced by an ethoxy group in position 2 (10m), selectivity increased significantly. Our research showed that the number of nitrogen atoms in the arylpiperazine system also strongly affected binding to the 5-HT receptors. When the ligand had two nitrogen atoms at positions 2 and 6, the activity was >100 nM for all receptors (10o). When there was only one nitrogen atom in the arylpiperazine system (10n), the activity with the 5-HT 1A and D 2 receptors increased significantly, after which, when the nitrogen atoms were completely eliminated (10p), the compound was active only with 5-HT 1A . Of all the trazodone derivatives obtained, the characteristics of 5-HT 1A receptor ligands were shown by 10e, 10g, 10h, 10i, 10m, and 10p. It is also worth noting that very interesting features as a dual 5-HT 1A / 7 ligand were shown by 10r.

Molecular Modeling
In order to explain the effect of the alkyl linker elongation in trazodone on the affinity to receptors 5-HT 1A and 5-HT 2A , we performed the molecular docking of the compound and representative ligand 10e (entry 6 in Table 6). The results showed that 10e, which contained the hexyl chain, did not bind to any used homology models of the serotonin receptor type 2A. In the case of the 5-HT 1A R, two alternative binding modes were considered (Figure 2). For both, the key salt bridge with D76 and protonated piperazine moiety was observed. In the case of bent conformations, on the left of Figure 2 the chlorophenyl ring of both compounds formed a CH-π interaction with F321/F322 and the 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one group interacted with N346. The main differences occurred in that last region. The elongated structure of 10e caused contacts with G342 and I345, while the trazodone interacted with T339 and A343. The second arrangement in consideration, which was more coherent, was additionally supported by the results of crystallographic studies-the crystal structure of trazodone hydrochloride, CSD-CPTAZP [25] (B on Figure 2). In this case, the triazolopyridine ring expanded into the cavity between transmembrane domains (TMs) 2 and 7. This binding mode meant that only trazodone could interact with phenylalanine F322 and W318, which was the main reason for its greater affinity in comparison to a hexyl derivative. In this case, the ligand with the hexyl chain additionally had contact with residues from ECL2 (Q57 and Y56) and from TM5 (S159).

Materials and Methods
The reactions in the microwave radiation field were mainly carried out in an Erlenmeyer flask in the Samsung M182DN device (300 W) and comparably in a closed PTFE tube in a Magnum II reactor (600 W) and CEM Discover SP reactor (100 W). All reagents from Sigma Aldrich (Poznan, Poland) and all organic solvents from POCH were of reagent grade and were used without purification. The progress of all reactions and purity of the synthesized compounds was confirmed by TLC, performed on Merck silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). Spots were detected by their absorption under ultraviolet (UV) light (λ = 254 nm). HPLC chromatograms were determined on a Perkin Elmer Series 200 HPLC with an XTerra RP C-18 (3.5 µm seed size, 4.6 × 150 mm) column and MeOH:H2O 1:1 eluent acidified with 0.1% formic acid as a phase (flow rate of 1 mL•min −1 ) was used. IR spectra were taken on an FTS-165 spectrometer (FTIR Biorad). Melting points were determined on a Boetius apparatus and are uncorrected. The purification by HPLC was assessed by comparing product integration to overall integrated spectrum. 1 H-NMR and 13 C-NMR spectra were recorded at 300 MHz (Bruker Avance, Cracow, Poland) using tetramethylsilane (TMS; 0.00 ppm) and chloroform-d1; J values are in Hertz (Hz), and splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), m (multiplet).
The three-dimensional structures of the ligands were fully optimized at CAM-B3LYP/6-31G*19 level with the polarizable continuum model (PCM) (solvent = water) using Gaussian 09 software (Gaussian, Inc., Wallingford, CT, USA). The appropriate ionization states at pH = 7.4 ± 1.0 were assigned using MarvinSketch 18.29 (ChemAxon Europe, Budapest, Hungary). The AutoDock Tools was used to assign the bond orders, appropriate amino acid ionization states, and to check for steric clashes. The receptor grid was generated by centering the grid box with a size of 12 Å on D76 side chain. Automated flexible docking was performed using AutoDock Vina 1.5.6 [26]. The figures were prepared using PYMOL.
The homology models of the selected serotonin receptors, namely 5-HT1A and 5-HT2A, were built

Materials and Methods
The reactions in the microwave radiation field were mainly carried out in an Erlenmeyer flask in the Samsung M182DN device (300 W) and comparably in a closed PTFE tube in a Magnum II reactor (600 W) and CEM Discover SP reactor (100 W). All reagents from Sigma Aldrich (Poznan, Poland) and all organic solvents from POCH were of reagent grade and were used without purification. The progress of all reactions and purity of the synthesized compounds was confirmed by TLC, performed on Merck silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany). Spots were detected by their absorption under ultraviolet (UV) light (λ = 254 nm). HPLC chromatograms were determined on a Perkin Elmer Series 200 HPLC with an XTerra RP C-18 (3.5 µm seed size, 4.6 × 150 mm) column and MeOH:H 2 O 1:1 eluent acidified with 0.1% formic acid as a phase (flow rate of 1 mL·min −1 ) was used. IR spectra were taken on an FTS-165 spectrometer (FTIR Biorad). Melting points were determined on a Boetius apparatus and are uncorrected. The purification by HPLC was assessed by comparing product integration to overall integrated spectrum. 1 H-NMR and 13 C-NMR spectra were recorded at 300 MHz (Bruker Avance, Cracow, Poland) using tetramethylsilane (TMS; 0.00 ppm) and chloroform-d1; J values are in Hertz (Hz), and splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), m (multiplet). The three-dimensional structures of the ligands were fully optimized at CAM-B3LYP/6-31G*19 level with the polarizable continuum model (PCM) (solvent = water) using Gaussian 09 software (Gaussian, Inc., Wallingford, CT, USA). The appropriate ionization states at pH = 7.4 ± 1.0 were assigned using MarvinSketch 18.29 (ChemAxon Europe, Budapest, Hungary). The AutoDock Tools was used to assign the bond orders, appropriate amino acid ionization states, and to check for steric clashes. The receptor grid was generated by centering the grid box with a size of 12 Å on D76 side chain. Automated flexible docking was performed using AutoDock Vina 1.5.6 [26]. The figures were prepared using PYMOL.
The homology models of the selected serotonin receptors, namely 5-HT 1A and 5-HT 2A , were built on the D 3 template (Protein Data Bank (PDB) identifier (ID): 3PBL), using a procedure described previously [27]. Firstly, 500 mg (4.58 mmol) g of 2-hydrazinopyridine (7) and 275 mg (4.58 mmol) or 550 mg (9.16 mmol) of urea were placed in a conical flask. Reactions were carried out under microwave radiation. The progress of the reaction was monitored by TLC (chloroform-methanol 9:1). After 50 s, 15 cm 3 of water was added to the mixture, after which the resulting product was filtered off on a Büchner funnel. The product was obtained with efficiency 49% in an equimolar reaction and 75% by reaction with a twofold molar excess of urea; R f = 0.62, HPLC 97.9% (reaction with molar excess), t M 1.95 min. Reactions were carried out under microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). After 2 min, the reaction mixture was cooled to about 60 • C and 15 cm 3 of water was added. The precipitate was filtered on a Büchner funnel and washed with about 50 cm 3 of water. Y = 48%.   (26 mmol, 4.09 g) of 1-bromo-3-chloropropane (2a)/2.65 cm 3 (0.026 mol, 5.25 g) of 1,3-dibromopropane (2b), 320 mg (0.001 mol) of TBAB, 4.14 g (0.03 mol) of K 2 CO 3 , and the appropriate amount of acetonitrile (6, 3, 0.75, or 0.4 cm 3 )/DMF (5 cm 3 ) were placed in a conical flask, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (chloroform-methanol 9:1). The reaction times are summarized in Table 1. After the reaction, about 50 cm 3 of water was added and the resulting product was filtered off on a Büchner funnel. The reaction yields are summarized in Table 1. The purity of the product obtained with the highest efficiency (Y = 92%) was confirmed using HPLC 99.3% t M = 3.43 min.  (1), 0.25 cm 3 (2.6 mol) of 1-bromo-3-chloropropane (2a), 32 mg (0.1 mmol) of TBAB, and 414 mg (3 mmol) of K 2 CO 3 0.075 cm 3 of acetonitrile were placed in a PTFE vessel, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). After 2 min, 5 cm 3 of water was added and the resulting product was filtered off on a Büchner funnel. Y = 81%.  (4), 320 mg (1 mmol) of TBAB, 4.14 g (30 mmol) of K 2 CO 3 , and the appropriate amount of acetonitrile (8, 3, or 1 cm 3 )/DMF (6, 4, or 2 cm 3 )/water (50 cm 3 ), were placed in a conical flask, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). The reaction times are summarized in Table 2. After the reaction, 50 cm 3 water was added and the resulting product was filtered off on a Büchner funnel. The reaction yields are summarized in Table 2. After drying, the obtained trazodone was dissolved in acetone and a solution of 2M HCl in dioxane was added until acidic (universal indicator). The precipitated hydrochloride was filtered off on a Büchner funnel.  Table 2. After drying, the obtained trazodone was dissolved in acetone and a solution of 2M HCl in dioxane was added until acidic (universal indicator). The precipitated hydrochloride was filtered off on a Büchner funnel.

General Procedures for the Preparation of 1-(3-Chloropropyl)-4-(3-chlorophenyl)piperazine (5)
3.6.1. Preparation of 1-(3-Chloropropyl)-4-(3-chlorophenyl)piperazine (5) under Microwave Radiation (Samsung M182DN; 300 W) Firstly, 2.50 cm 3 (26 mmol, 4.09 g) of 1-bromo-3-chloropropane (2a), 2.33 g (10 mmol) of 1-(3-chlorophenyl)piperazine hydrochloride (4), 320 mg (10 mmol) of TBAB, 4.14 g (30 mmol) of K 2 CO 3 , and 3 cm 3 of acetonitrile/DMF were placed in a conical flask, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). After the reaction, 50 cm 3 of water was added and the resulting product was filtered off. After drying, obtained 5 was dissolved in acetone and a solution of 2M HCl in dioxane was added until acidic (universal indicator). The precipitated hydrochloride was filtered off on a Büchner funnel; R f = 0.83. The reaction yields are summarized in Table 3.  (4), 32 mg (1 mmol) of TBAB, 414 mg of K 2 CO 3 (3 mmol), and 0.3 cm 3 of acetonitrile were added in a PTFE vessel, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). After drying, obtained 5 was dissolved in acetone and a solution of 2M HCl in dioxane was added until acidic (universal indicator). The precipitated hydrochloride was filtered off on a Büchner funnel.  (1), 322 mg (1 mmol) of TBAB, 4.14 g (30 mmol) of K 2 CO 3 , and a corresponding amount of acetonitrile (2, 6, or 8 cm 3 ) were placed in a conical flask, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). The reaction times are summarized in Table 4. After the reaction, about 50 cm 3 of water was added and the resulting product was filtered off on a Büchner funnel. The reaction yields are summarized in Table 4; R f = 0.75.  (1), 32 mg (0.1 mmol) of TBAB, 414 mg (3 mmol) of K 2 CO 3 , and 0.2 cm 3 of acetonitrile were placed in a PTFE vessel, after which the mixture was subjected to microwave radiation. The progress of the reaction was monitored by TLC (eluent chloroform-methanol 9:1). After the reaction, water was added and the resulting product was filtered off. After drying to solution 1-(3-chloropropyl)-4-(3-chloropheyl)piperazine (5) in acetone, a solution of HCl in dioxane was added until acidic. The precipitated hydrochloride was filtered off on a Büchner funnel.