Synthesis and Structure-Activity Relationship Analysis of 5-HT7 Receptor Antagonists: Piperazin-1-yl Substituted Unfused Heterobiaryls

A series of piperazin-1-yl substituted unfused heterobiaryls was synthesized as ligands of the 5-HT7 receptors. The goal of this project was to elucidate the structural features that affect the 5-HT7 binding affinity of this class of compounds represented by the model ligand 4-(3-furyl)-2-(4-methylpiperazin-1-yl)pyrimidine (2). The SAR studies included systematical structural changes of the pyrimidine core moiety in 2 to quinazoline, pyridine and benzene, changes of the 3-furyl group to other heteroaryl substituents, the presence of various analogs of the 4-methylpiperazin-1-yl group, as well as additional substitutions at positions 5 and 6 of the pyrimidine. Substitution of position 6 of the pyrimidine in the model ligand with an alkyl group results in a substantial increase of the binding affinity (note a change in position numbers due to the nomenclature rules). It was also demonstrated that 4-(3-furyl) moiety is crucial for the 5-HT7 binding affinity of the substituted pyrimidines, although, the pyrimidine core can be replaced with a pyridine ring without a dramatic loss of the binding affinity. The selected ethylpyrimidine (12) and butylpyrimidine (13) analogs of high 5-HT7 binding affinity showed antagonistic properties in cAMP functional test and varied selectivity profile—compound 12 can be regarded as a dual 5-HT7/5-HT2AR ligand, and 13 as a multi-receptor (5-HT7, 5-HT2A, 5-HT6 and D2) agent.


Introduction
For more than 60 years, the studies on the role of serotonin (5-HT) within the central nervous system (CNS) have constantly provided evidence of the involvement of 5-HT receptors in the action of various psychiatric drugs. It is now evident that deregulation of serotonergic signaling is associated with pathogenesis of severe neuropsychological disorders like depression [1,2] Alzheimer's The classical alkylation and acylation reactions of 1 provided facile entry to compounds 3-10 (Scheme 2). A similar strategy afforded a piperazinium derivative 47 (see Experimental). It was also found that the conjugate addition reaction of dimethylamine with 2-chloro-4-vinylquinazoline [27,28] proceeds cleanly to furnish 2-chloro-4-(2-dimethylamino)ethyl substituted quinazoline, the subsequent treatment of which with N-methylpiperazine yielded the desired product 50 (Scheme 5).

57-59
Scheme 8. Synthesis of substituted pyridines 57-59 (see Table 4).  Table 4). In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Scheme 9. Synthesis of substituted benzenes 60-71 (see Table 5).  In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5).  In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5). In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5). In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5).  In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5).  In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5). In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5).  In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.  Table 5). In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.
In case of benzene derivatives, Buchwald-Hartwig amination of halogeno-substituted benzenes with N-methylpiperazine in the presence of a palladium catalyst provided piperazine-substituted products (Scheme 9). The Suzuki condensation of these intermediate products with heteroarylboronic acids yielded the desired heteroaryl-substituted benzenes 60-71. It should be noted that the majority of the two-step reactions discussed above were conducted with crude intermediate products. However, the final products were rigorously characterized in all cases studied.

Pharmacology
Novel scaffold endowed with 5-HT 7 R activity was identified by screening of the in-house compound library in a single compound concentration of 1 µM in a competition radioligand binding assay using [ 3 H]5-CT and HEK293 cells overexpressing the human 5-HT 7b receptors. Compounds showing >60% of inhibition, namely (4-(3-furyl)-2-(4-methylpiperazin-1-yl)pyrimidine (2) and its two derivatives 11 and 18 were further tested in full binding experiments, and, as for all the newly synthesized compounds (1, 3-10, 12-17 and 19-71), 5-HT 7 R affinity (K i values˘SD) was determined in at least two independent experiments run in triplicate [31]. The functional properties of two selected compounds 12 and 13 active on 5-HT 7 R were further evaluated using their ability to inhibit cAMP production induced by 5-CT (10 nM) in the same cellular system [32,33]. In addition, compounds 12 and 13 were evaluated for their affinity at three other serotonergic receptors: 5-HT 1A , 5-HT 2A , 5-HT 6 and dopaminergic D 2 in standard competition binding assays by use of the appropriate radioligand:

Discussion
The objective of this study was to determine which combinations of structural features affect the 5-HT 7 binding affinity of unfused heterobiaryls. The intended SAR studies included synthesis of new analogs of the model compound 2 and encompassed the following points: (1) substitution of the amino group within the piperazine ring; (2) introduction of alkyl, amino and bulky substituents into positions 4/6 and/or 5 of the pyrimidine ring; (3) modifications of the heteroaryl group in position 4/6 of the pyrimidine; (4) replacement of the piperazinyl group by other amino substituents; and (5) synthesis of a series of pyridines and benzenes allowing comparison of the influence of the central ring nitrogen atom(s) on the ligand affinity.
First, investigating the influence of substituents at the basic piperazine atom, it was found that unsubstituted (1), simple alkyls (3 and 4), arylalkyl (7), hydroxyalkyl (8) or even esters (9 and 10) provide a similar level of 5-HT 7 R affinity (K i = 1.4-10 nM) as methylated hit compound 2 (K i = 7.2 nM). Slight decrease in 5-HT 7 R affinity is observed for benzyl derivative 5 (K i = 25 nM) which is probably caused by a negative steric influence of the phenyl group on the key interaction between a protonated nitrogen and aspartic acid (D3.32) residue in the receptor [35]. Moreover, with the benzoyl group (6), there is an obvious negative effect on the affinity (K i = 1405 nM) that arises from diminished basicity of the piperazine nitrogen atom (Table 1).

± 42
Next it was found that quinazoline derivatives (48-50), which can be treated as 5,6-disubstituted pyrimidine analogs, were inactive (Ki > 1910 nM). This result is fully consistent with our finding that substitution at position 5 is highly detrimental for 5-HT7R affinity (Table 3). Having established reasonable SAR around pyrimidine central ring, we focused our attention on the role of the pyrimidine core itself, analyzing a series of substituted pyridine-and phenyl-piperazine analogs. The pyridine derivatives (51-59) were then synthesized to determine whether both nitrogen atoms within the central heterocycle are needed for the ligand to retain affinity for the binding pocket of the receptor ( Table 4). The biological data show that both isomeric 4-(3-furyl) (51) and 6-(3-furyl) (57) pyridine ligands still demonstrate high affinities for the 5-HT7R (Ki = 17 nM and 8 nM, respectively). However, for the 2-furyl derivatives, position 4 (52 Ki = 307 nM) is preferred over Next, the SAR analysis of the substituents at position 4 of the pyrimidine ring was investigated by modifications and replacement of the 3-furyl group. Compounds 23, 24 with methyl substituents within the 3-furyl ring display reduced affinities (K i values of 1,920 and 462 nM, respectively). Interestingly, introduction of an n-hexyl group (25) at position 6 of the pyrimidine in compound 24 restores high affinity for 5-HT 7 R (K i = 12 nM). A similar trend can be recognized for less active ligands 26 (2-furyl, K i = 1,021 nM) and 30 (3-thienyl, K i = 83 nM), where addition of a 6-alkyl substituent results in a 2-or 3-fold boost in affinity (ethyl, 27, K i = 380 nM; n-butyl, 28, K i = 450 nM and n-butyl, 31, K i = 15 nM). As a general rule, 3-furyl (2, 24) and 3-thienyl (30) derivatives are 5 to 140-fold more active than their 2-furyl (26, 29) and 2-thienyl (32) counterparts. Replacement of the 3-furyl group for o-, m-, p-hydroxyphenyl (33-35), pyridin-3-yl (39), thiazol-2-yl (40) or imidazol-1-yl (41) group as well as benzo-fused heterocyclic (36-38) moieties does not furnish ligands with noteworthy affinity for 5-HT 7 R (K i > 1000 nM). Thus, it appears that the 3-furyl group is necessary for a high affinity of 5-HT 7 R ligands or, alternatively, a 3-thienyl moiety but with an appropriate enhancing substitutent at position 6 of the pyrimidine ring (Table 1).
Next it was found that quinazoline derivatives (48-50), which can be treated as 5,6-disubstituted pyrimidine analogs, were inactive (K i > 1910 nM). This result is fully consistent with our finding that substitution at position 5 is highly detrimental for 5-HT 7 R affinity (Table 3).

± 42
Next it was found that quinazoline derivatives (48-50), which can be treated as 5,6-disubstituted pyrimidine analogs, were inactive (Ki > 1910 nM). This result is fully consistent with our finding that substitution at position 5 is highly detrimental for 5-HT7R affinity (Table 3). Having established reasonable SAR around pyrimidine central ring, we focused our attention on the role of the pyrimidine core itself, analyzing a series of substituted pyridine-and phenyl-piperazine analogs. The pyridine derivatives (51-59) were then synthesized to determine whether both nitrogen atoms within the central heterocycle are needed for the ligand to retain affinity for the binding pocket of the receptor ( Table 4). The biological data show that both isomeric 4-(3-furyl) (51) and 6-(3-furyl) (57) pyridine ligands still demonstrate high affinities for the 5-HT7R (Ki = 17 nM and 8 nM, respectively). However, for the 2-furyl derivatives, position 4 (52 Ki = 307 nM) is preferred over Next it was found that quinazoline derivatives (48-50), which can be treated as 5,6-disubstituted pyrimidine analogs, were inactive (Ki > 1910 nM). This result is fully consistent with our finding that substitution at position 5 is highly detrimental for 5-HT7R affinity (Table 3). Having established reasonable SAR around pyrimidine central ring, we focused our attention on the role of the pyrimidine core itself, analyzing a series of substituted pyridine-and phenyl-piperazine analogs. The pyridine derivatives (51-59) were then synthesized to determine whether both nitrogen atoms within the central heterocycle are needed for the ligand to retain affinity for the binding pocket of the receptor ( Table 4). The biological data show that both isomeric 4-(3-furyl) (51) and 6-(3-furyl) (57) pyridine ligands still demonstrate high affinities for the 5-HT7R (Ki = 17 nM and 8 nM, respectively). However, for the 2-furyl derivatives, position 4 (52 Ki = 307 nM) is preferred over 7540˘1140

General Methods
All air-sensitive reactions were conducted under a nitrogen atmosphere. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl immediately before use. Heteroaryllithium

General Methods
All air-sensitive reactions were conducted under a nitrogen atmosphere. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl immediately before use. Heteroaryllithium reagents were generated by direct lithiation of a heterocyclic substrate or by a bromine-lithium exchange reaction as previously reported [28] Final products were purified on a chromatotron using silica gel-coated rotors. Oily products were transformed into hydrobromide salts by using a general procedure [30] and the salts were crystallized from 95% ethanol. Melting points (Pyrex capillary) are not corrected. The 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra of free bases and hydrobromide salts were obtained in CDCl 3 and DMSO-d 6 , respectively. Mass spectra were recorded using electrospray ionization in a positive ion mode. Substituted Pyrimidines 1, 2, 21, 23, 24, 26, 29, 30, 32,
The conjugate addition reaction of dimethylamine with 2-chloro-4-vinylquinazoline to give 2-chloro-4-(2-dimethylaminoethyl)quinazoline was conducted as described for the preparation of analogous compounds [27]. Treatment of this compound with 4-methylpiperazine to give the final product 50 was conducted by using a general procedure described above.