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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Bioorg Med Chem. 2014 Apr 20;22(12):3105–3114. doi: 10.1016/j.bmc.2014.04.026

Identification of a New Selective Dopamine D4 Receptor Ligand

Dinithia Sampson a,#, Xue Y Zhu a,#, Suresh V K Eyunni a, Jagan R Etukala a, Edward Ofori a, Barbara Bricker a, Nazarius S Lamango a, Vincent Setola b, Bryan L Roth b, Seth Y Ablordeppey a,*
PMCID: PMC4096627  NIHMSID: NIHMS592404  PMID: 24800940

Abstract

The dopamine D4 receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D4 ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD4 receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D4 receptor. Compound 27 (KiD4 = 0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D4 receptor. Compound 28 (KiD4 = 3.9 nM) while not as potent, was more discriminatory for the D4 receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT1AR and 5HT2BR, have binding affinity constants better than 100 nM (Ki < 100 nM). Compound 28 is a potentially useful D4-selective ligand for probing disease treatments involving the D4 receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted.

1. Introduction

Targeting dopamine receptors for drug development has been of interest for decades because of their potential utility in many well-known pathological conditions.1 Initially, dopamine receptors were classified as D1-like and D2-like for many years until it became evident that the D1-like receptors consisted of D1 and D5 subtypes and the D2-like receptors consisted of D2, D3 and D4 subtypes. By far, the D2-like receptors have been studied more because of their association with clinically relevant neuropsychiatric conditions, such as schizophrenia, mood disorders, Parkinson’s disease and others. Each of these subtypes is now separately associated with certain pathophysiological conditions. For example, several D2 agonists and partial agonists have shown beneficial effects in counteracting Parkinson’s disease, attention-deficit hyperactivity disorder and certain mood disorders2 while D2 antagonism is associated with antipsychotic properties.

Since its cloning, the D4 receptor has attracted considerable interest. For example, early reports indicated that D4 antagonists attenuate not only the discriminative-stimulus effects of cocaine and methamphetamine3–4 but also morphine-induced withdrawal signs induced by naloxone.5 More recently, there was a report that the D4 receptor antagonist, L-745,870 (Chart 1) but not PD 168,077, a selective D4 receptor agonist, attenuated nicotine-induced reinstatement of nicotine seeking behavior.6 Thus, it would appear that the pharmacological blockade of DAD4 may serve as a new and potentially effective treatment against relapse to tobacco smoking. On the other hand, PD 168,077 has been shown to induce penile erection in rats when administered in vivo, and L-745,870 was able to block this action, and thus, confirming the D4 receptor mediated mechanism by which penile erection occured.7 This potential could place D4 agonists in a strong position to replace the current PDE5 antagonists with a plethora of side-effects.8 In addition, D4-receptor agonists may be useful in reversing cognitive deficits in schizophrenia.9 These demonstrated therapeutic potentials have encouraged the search for new D4 selective ligands in our laboratories.

Chart 1.

Chart 1

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Known D4 selective Ligands with a Piperazine Pharmacophore

We have previously carried out several SAR studies that sought to identify structural entities that demonstrate DA receptor subtype selectivity.10–12 A frequent observation in such studies was the fact that unlike the piperidine analogs of haloperidol, the piperazine analogs demonstrated selective and significant affinities to the D4 receptor subtype. Chart 1 displays common D4 selective ligands with the piperazine pharmacophore. The purpose of the current study was to further explore the piperazine ring as a pharmacophore in a search for new entities that are selective to the D4 receptor subtype using compound 1 (Chart 1), the piperazine analog of haloperidol as the lead.

2. Chemistry

The syntheses of compounds 1–7 (Chart 2) have previously been reported using routine N-alkylation reactions.9 Compound 8, the cyclic analog of 1, was synthesized as depicted in Scheme 1 below. The commercially available 4-chloro-1-(4-fluorophenyl)-butan-1-one (29) was converted to 4-chloro-1-(4-fluorophenyl)-2-methylenebutan-1-one (30) by refluxing in acetic anhydride in the presence of hexamethylenetriamine.10,13 Compound 30 was cyclized by heating in concentrated sulfuric acid to produce 2-(2-chloroethyl)-5-fluoro-2,3-dihydro-1H-inden-1-one (31) which was protected by reaction with ethylene glycol to form the 1,3-dioxolane, 32. Alkylating 4-(4-chlorophenyl)piperazine with 32 and deprotecting the dioxolane resulted in the formation of the desired target compound 8 (Scheme 1).

Chart 2.

Chart 2

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4-Chlorophenyl piperazine analogs

Scheme 1. Synthesis of 4-chlorophenylpiperazine analogs, 8–10.

Scheme 1

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Reagents and conditions: i) HMTA, Ac2O, Reflux; ii) Conc. H2SO4, 60°C; iii) a) Ethylene glycol, TsOH, Reflux, 48 h; b) NaBH4, MeOH; iv) Zn/HgCl2, Conc. HCl, Toluene; v) KI, K2CO3, DME, 90°C, 4-(4-chlorophenyl)piperazine, 12 h; vi) TsOH, MeOH, rt

Compound 9 was similarly obtained using the desfluoro starting material 4-chloro-1-(phenyl)butan-1-one. To obtain compound 10, the indanone 31 was deoxygenated (32) under Clemmensen reaction conditions14 and then used to alkylate 4-(4-chlorophenyl)piperazine (Scheme 1).

Compounds 11–14 and 16a–c (Chart 3) were previously synthesized as a part of a drug design effort to obtain novel antipsychotic drugs.9 In general, an appropriately substituted phenol or benzenethiol (34) was alkylated using 3-chloropropan-1-ol (35) and the resulting alcohol (36) was either tosylated or mesylated and then utilized in alkylating the heteroaryl piperazine (A–C) in Scheme 2. The synthesis of compounds 15–16 followed the same procedure reported in Scheme 2.

Chart 3.

Chart 3

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Heteroaryl piperazine analogs.

Scheme 2. Synthesis of heteroaryl piperazine analogs. 11–16.

Scheme 2

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Reagents and conditions: i) KI, K2CO3, iPrOH or DME, Reflux; ii) MsCl/TsCl, Et3N, DCM, rt; iii) KI, K2CO3, DME, 90°C, heteroaryl piperazine, 12 h.

The syntheses of compounds 17–18 (Chart 4) were also previously reported.15 To obtain the benzo[d]oxazole analogs, 19–22 (Chart 4, Scheme 3), 2-aminophenol was reacted with chloroalkanoyl alkyl halide (39) to form the amide intermediate (40) which was then cyclized using polyphosphoric acid (PPA) to obtain 2-(2-chloroalkyl)benzo[d]oxazole, 41. Compound 41 was used to alkylate 4-chlorophenyl-1-piperazine to yield the desired target compounds 19–22.

Chart 4.

Chart 4

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Benzothiazole, Benzoxazole and Benzofuran Analogs

Scheme 3. Synthesis of benzoxazole derivatives, 19–22.

Scheme 3

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Reagents and Conditions: (i) EtOAc, TEA, Reflux; (ii) PPA, 110°C; (iii) CH3CN, K2CO3, KI, Reflux.

The synthesis of benzofuran moiety in analogs 23–26 (Chart 4), followed a literature procedure.16 2-Iodophenol was reacted with an appropriate alkyl-1-yn-1-ol to form the corresponding benzofuran alkanol, which was subjected to a tosylation reaction and the resulting product was used to alkylate an appropriate aryl piperazine or a related analog to form the target compounds as shown in Scheme 4.

Scheme 4. Synthesis of benzofuran analogs, 23–26.

Scheme 4

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Reagents and conditions: (i) An appropriate alkyl-1-yn-1-ol, Cu2O, pyridine, 100°C, 15h (ii) TosylCl, NEt3, CH2Cl2, rt, overnight; (iii) NHR2 [Aryl piperazine or related analog], ACN, NEt3, 60°C, reflux.

Finally, the synthesis of compound 28 (Chart 4) followed the synthetic procedure previously utilized in obtaining compound 27 (Chart 4) as shown in Scheme 5.15 Briefly, 2-aminobenzenethiol (46) was reacted with 4-chlorobutanoyl chloride (47) to form 2-(3-chloropropyl)-benzo[d]thiazole, 48, which was then used to alkylate 2-(piperazin-1-yl)pyrimidine to form the desired target compound, 28.

Scheme 5. Synthesis of benzothiazole analog, 28.

Scheme 5

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Reagents and conditions: (i) Toluene, rt; (ii) KI, K2CO3, CH3CN, reflux.

3. Results and Discussion

Cigarette smoking is associated with major diseases including cardiovascular problems, stroke and cancers of several organs. In fact, according to the CDC website, cigarette smoking harms nearly every organ of the body and affects a person’s overall health.17 Quitting smoking cuts down on all risks including, cardiovascular risks, stroke, and cancers of the lung, mouth, throat, esophagus and bladder.18 Unfortunately, smoking cessation is difficult and often requires therapeutic interventions. And yet there have been articles suggesting significant risks associated with some of the current pharmacological interventions.19 Thus, a search to find new agents that could help those addicted to cigarette smoking to quit the habit is an urgent and important public health need. Similarly, the discovery of the selective D4 agonist, PD168,077 as having the capability of reversing cognitive deficits in schizophrenia9 and inducing penile erection7 and hence the potential to treat erectile dysfunction is very interesting and could provide an alternative treatment option in place of the PDE5 antagonists with several known side-effects.8

Our laboratory’s attention has been drawn to these DAD4 receptor-mediated pathologies and that has spurred us to mine our databases for new pharmacophores for the D4 receptor. Previous publications from our lab have identified compounds 1–7 as having low affinities for the D2 receptor while exhibiting selectivity for the D4 receptor among D2-like receptors. We have now screened these compounds at the D1-like receptors (Table 1) and the results further demonstrate that their selectivity extends beyond the D2-like receptors and hence we selected compound 1 as the lead agent to conduct a structure-activity relationship (SAR) study that has led to the identification of potent and selective D4 ligands. Replacing the carbonyl moiety in compound 1 with a sulfoxide (2), methylene (3), sulfur (4) or oxygen (5) group/atom resulted in increasing potency at the D4 receptor with Ki values from 17.5 nM to 6.9 nM respectively while binding at other DA receptors showed no observable trends. The sulfoxide analog resulted in the lowest affinity binding at the rest of the DA receptors while its reduced counterpart, the sulfide (4) had moderate binding except at the D1 receptor where its affinity binding Ki was 52 nM.

Table 1.

Evaluation of binding affinities of 4-chlorophenyl analogs (Chart 2) at the Dopamine Receptor subtypes

Compd Ki (pKi)±SEM values in nM at the Dopamine Receptor Subtypes
D1 D2 D3 D4 D5 D2/D4 D3/D4
1. ND 253.3±38.9 403.9±66 17.5±2.0 ND 14.5 23.1
2. MT 635(6.2±0.05) 1340(5.87±0.06) 13(7.89±0.05) MT 48.8 103
3. 589±56 284±21 261±23 7.8±0.4 1758±212 36.4 33.5
4. 52±3 211±22 422±41 8.7±0.5 2808±325 24.3 48.5
5. 135±8 390±34 885±65 6.9±0.3 2684±223 56.5 128
6. ND 447(6.35±0.07) 726(6.1±0.1) 5.6 (8.26±0.03) ND 79.8 130
7. 126±9 41.0±4.0 696±50.0 9.5±0.3 867±49 4.3 73.3
8. 641(6.19±0.06) 1543(5.81±0.08) 2477(5.6±0.1) 6.1(8.21±0.05) MT 253 406
9. 1701 (6.41±0.07) >10,000 4534 (5.34±0.08) 36.0 (7.45±0.04) MT >277 126
10. 837±92 1417±138 2772±271 13.0±1.0 MT 109 213
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MT = Missed primary assay threshold of 50% inhibition; ND = Not determined

Overall, these compounds have demonstrated better selectivity toward the D4 receptor when compared to compound 1. In particular, compound 5, the oxygen analog, not only had the best binding at the D4 receptor, but demonstrated the highest selectivity among compounds 1–5. Further probing of the oxygen analog revealed that removing the para fluoro atom (6) has little or no contribution to binding to the D2-like receptors while replacement of N-4 in the piperazine ring with CH (7) resulted in a very significant increase in potency for the D2 receptor with only minimal changes elsewhere. Compound 8 was designed and synthesized to explore the effect of restricting the carbonyl group in an indanone ring, on binding affinity and selectivity. Indeed, this transformation resulted in one of the highest selectivities toward the D4 receptor; the ratios for the D2/D4 and D3/D4 being 253 and 406 respectively. Synthesis of 9, an analog of 8 but without the fluorine atom resulted in a significant decrease in binding at all the DA subtypes including the D4 suggesting the fluorine may have some important interaction at the binding sites for these indanones. Deoxygenation of the carbonyl to form compound 10 however, produced only minimal changes.

The next evaluations focused on the replacement of the 4-chlorophenyl moiety. Our previous work indicated that pyridine and pyrimidine rings impacted binding to CNS receptors.9–12 Hence, we first explored replacing the 4-chlorophenyl moiety with the pyridine ring in compounds 3–5 to obtain compounds 11–13 (Chart 2) and the binding affinities are reported in Table 2. Compounds 11–13 bind with very high affinities at D4 versus D1–D3 receptors, and therefore demonstrate decreased selectivity for the D4 receptor. Compound 14, an analog of 13 with the fluorine atom replaced with a trifluoromethyl group, displayed significantly lower binding affinities for all DA receptors suggesting either a steric limitation or deleterious electron withdrawing effect or both. Replacement of the pyridine ring with 5-methyl substituted pyridine for the trio (11–13) to form compounds 15a–c were also evaluated. Interestingly, compound 15a with the oxygen linker resulted in the most potent analog for the D4 receptor (Ki = 1.1 nM) among the 17 compounds evaluated thus far. In addition, it also displayed the highest selectivity for the D4 receptor when compared with the D2 receptor. Not surprisingly, compounds 15b and 15c, the sulfur and carbon analogs respectively, have reduced binding for the D4 receptor but retain similar affinities for the D1–D3 receptors.

Table 2.

Evaluation of the binding affinities of heteroaryl piperazine analogs (Chart 3) at the DAR Subtypes

Compd Ki (pKi)±SEM values in nM at the Dopamine Receptor Subtypes
D1 D2 D3 D4 D5 D2/D4 D3/D4
11. 238±24.0 124±10.0 86±4.0 3.5±0.2 1451±133 35.4 24.6
12. 265±24.0 183±21.0 160±16.0 5.7±0.3 3223±295 32.1 28.1
13. 180±14.0 186±16.0 229±20.0 1.8±0.1 2392±252 103 127
14. 2344 (5.63±0.09) 1092 (5.96±0.09) 355 (6.45±0.05) 12 (7.9±0.1) >10,000 91.0 29.6
15a. 260 (6.58±0.07) 1046 (5.98±0.07) 187 (6.7±0.1) 1.1 (8.96±0.08) 2576 (5.6±0.1) 951 170
15b. 222 (6.65±0.07) 1106 (6±0.1) 170 (6.8±0.1) 20.0 (7.71±0.06) 2159 (5.7±0.1) 55.3 8.5
15c. 521 (6.28±0.08) 1861 (5.7±0.1) 156 (6.8±0.1) 23.0 (7.64±0.06) 3264 (5.5±0.1) 80.9 6.8
16a. 259±12.0 636±66.0 778±63.0 4.2±0.1 MT 151 185
16b. 495±33.0 424±25.0 68.0±10.0 21.0±1.0 1370±114 20.1 3.2
16c. 134±7.0 269±17.0 262±17.0 6.2±0.3 3812±409 43.4 42.3
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MT = Missed primary assay threshold of 50% inhibition; ND = Not determined

Finally in this series, compounds 16a–c, the pyrimidine analogs of 11–13 were synthesized and evaluated. Once again the oxygen analog 16a displayed the highest potency and selectivity for the D4R among the three analogs. The carbon analog (16c) had a similar binding profile at the DA subtypes as 16a although its selectivity for the D4R is much lower. Meanwhile, the sulfur analog 16b again demonstrated a 5-fold lower affinity for the D4R compared to 16a. These evaluations have clearly demonstrated that the oxygen analogs have the highest potencies for the D4R within each cohort evaluated. Comparing the pyridine and pyrimidine analogs in this series, the pyridine analogs overall, demonstrated moderate enhancement in binding affinity at the D4R than the pyrimidine analogs. In addition, among the twenty compounds evaluated, none has a better binding affinity for the D5 receptor than compound 7 with a Ki of 867 nM. In other words, the compounds have little or no affinity for the D5R.

The last group of compounds evaluated is 17 – 28 (Chart 4), which may be considered as the heterobicyclic analogs of the compounds in charts 2 and 3, with binding affinities reported in Table 3. Compound 17 and 18 are benzothiazole analogs which were previously synthesized and evaluated for binding to the D2-like receptors.15 In this paper, their binding affinities to the D1-like receptors were evaluated and the results are reported. The results suggest that a chain length of 4 (17) produced a more potent agent at the D4R than a chain length of 3 (18). The selectivities of both compounds for the D4R were unremarkable.

Table 3.

Evaluation of the binding affinities of benzothiazole, benzoxazole and benzofuran analogs (Chart 4) at the DAR subtypes

Compound Ki (pKi)±SEM values in nM at the Dopamine Receptor Subtypes
D1 D2 D3 D4 D5 D2/D4 D3/D4
17. 1897±364 219±12.0 31±4.0 4.0±0.0 ND 54.8 7.8
18. 1273±123 2321±314 214±47 31±40 >10,000 74.9 6.9
19. 7939±592 3108±1071 119±25 240±36.8 >10,000 13.0 0.5
20. MT MT 33±6 30.6±6.3 MT >327 1.1
21. 1172±76 3962±501 180±31 145±18 >10,000 27.3 1.5
22. MT MT 850±126 1409±227 MT >7.1 0.6
23. MT MT MT 282.9 MT >35.5- >35.5
24. 1408±242 1979±291 2290±423 78.9±5.7 MT 25.1 29.0
25. MT MT 670.7±118.4 2397±294 MT >4.2- 0.3
26. 274±24 2507±208 1462±133 185±11 1522±191 13.5 7.9
27. MT 26.5±4.5 100 (7±0.06) 0.84±0.09 MT 31.5 119
28. MT MT MT 3.9 (8.41±0.04) MT >2564 >2564
FAC 113a ND 3200±58 5000±121 3.1±0.3 ND
L-745870b ND 960 2300 0.43 ND
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MT = Missed primary assay threshold of 50% inhibition; ND = Not determined.

a

Ref 20;

b

Ref 21.

Next, we synthesized and evaluated the benzoxazole analogs, 19 – 22, by systematically modifying the chain length from 5 to 2 respectively. A chain length of five atoms (19) produced a weak binding affinity at the D4R (Ki = 240 nM) while 20, with a chain length of 4 atoms produced an 8-fold increase in binding to the D4 receptor (Ki = 30.6 nM), thus suggesting a chain length of 4 is preferred. Compound 20 however, has similar affinity for the D3R (Ki = 33 nM) resulting in loss of selectivity. Further comparison of this benzothiazole, 20 with the benzoxazole, 17 shows over 7-fold differential, suggesting the benzothiazole ring with a 4-methylene chain is preferred at the D4R. There is however no preference at the D3 receptor since they (17 & 20) have similar binding affinities. Compounds 21 and 22, benzoxazoles with a chain length of 3 and 2 respectively, resulted in compounds with only moderate to very weak binding affinities at all the DA receptors.

We also synthesized and evaluated compounds obtained by replacement of the benzoxazole with a benzofuran, (Chart 4), 23 – 26, of which 24 may be considered as a restricted analog of the straight chain ether analog in Chart 2. The results indicated that while somewhat selective for the D4 receptor, the 2 carbon chain analog (23) and the 3-chain analog (24) have weaker binding affinities for the D4 receptor. Replacement of the piperazine ring with its bridged counterpart (25) and with a homopiperazine (26) did not improve potency or selectivity.

Returning to the benzothiazoles, we further explored replacement of the 4-chlorophenyl moiety with 2-(piperazin-1-yl)pyrimidine to obtain the 4-chain (27) and 3-chain (28) analogs. Compound 27 was the most potent D4 ligand in this study (Ki = 0.84 nM) as previously reported15 albeit with diminished selectivity, while 28 has high potency (Ki = 3.9 nM) and is by far the most selective analog at the D4 receptor when compared with other dopamine subtypes. Taking their binding constants at face value, compound 28 is as potent but more selective than FAUC 113, a previously reported D4 ligand (Chart 1).20 In addition, while not as potent as L-745,860,21 compound 28 is also more selective for the D4 receptor among the D2-like receptors. To determine the extent of compound 28’s selectivity, we screened its binding affinities at several other CNS receptors and the results are reported in Table 4.

Table 4.

Binding Affinity at Other Relevant CNS Receptors, Ki in nM (pKi) ±SEM

Compd 5HT1A 5HT2A 5HT2B 5HT2C 5HT3 5HT6 5HT7 H1
28. 40.0
(7.39±0.04)		 283
(6.55±0.03)		 47.0
(7.33±0.05)		 280
(6.55±0.04)		 MT MT 342
(6.47±0.06)		 730
(6.14±0.06)
Compd α1A α2A α2C M1 M3 DAT NET SERT Sigma 1 Sigma 2
28. 259
(6.59±0.08)		 844
(6.07±0.04)		 126
(6.9±0.04)		 MT MT MT 2773
(5.56±0.06)		 MT 350
(6.46±0.05)		 162
(6.79±0.08)
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MT = Missed primary assay threshold of 50% inhibition

Screening experiments involving a total of 18 other receptors indicated that apart from the 5HT1A and 5HT2B receptors, where binding affinities were below 100 nM (Ki < 100 nM), compound 28 exhibited significantly poorer affinities for the remainder of the assayed receptors. The above results strongly suggest that compound 28 can be a potentially useful D4-selective ligand for probing D4R related pathophysiological conditions including smoking cessation, erectile dysfunction and reversal of cognitive deficits in schizophrenia depending on the intrinsic activity.

3.1. Conclusions

This study was initiated to identify selective DAD4 receptor ligands. The results confirm the piperazine ring as a reliable pharmacophore impacting potency and selectivity for the D4 receptor. Of the 25 piperazine derivatives evaluated, all displayed higher potencies at the D4 than at the D2 receptors. Compound 27 (Ki = 0.84 nM) has the highest potency at the D4 receptor but displays only moderate selectivity compared to the other DA subtypes. The most significant finding however is the identification of a novel benzothiazole alkyl piperazine, compound 28, with a binding affinity constant (Ki) of 3.9 nM and no significant binding affinity to any of the other DA receptor subtypes (less than 50% inhibition of the appropriate radioligand at each of the other DA subtypes). In addition, compound 28 has only weak to moderate affinities for eighteen other CNS receptors. These results warrant a more elaborate pharmacological profiling, including functional characterization, which is the focus of our current ongoing studies.

4. Experimental

Melting points were determined on a Gallenkamp (UK) apparatus and are uncorrected. All NMR spectra were obtained on a Varian 300 MHz Mercury Spectrometer. Elemental analyses were carried out by Atlantic Microlab, Inc., Norcross, GA, and are within 0.4% of theory unless otherwise noted. Flash chromatography was performed with Davisil grade 634 silica gel. Starting materials were obtained from Sigma-Aldrich and were used without further purification.

4.1. Synthetic Procedure

4.1.1. Synthesis of 2-(2-chloroethyl)-5-fluoro-indan-1-one, 31

A mixture of 4-chloro-1-(4-fluorophenyl)-butan-1-one, 29 (10 g, 50 mmol), hexamethylenetriamine (10.5 g, 75 mmol) in Ac2O (25 mL) was refluxed under N2 for 16 h. After cooling to rt, the mixture was diluted with CHCl3 (500 mL) and then washed with HCl solution (10%, 2×300 mL), H2O (300 mL), and sat NaHCO3 (300 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo, followed by column chromatography on silica gel to afford 4-chloro-1-(4-fluoro-phenyl)-2-methylene-butan-1-one, 30 (2.8 g, 26.4%). 1H NMR (CDCl3): δ 7.82 (dd, J = 9.0, 5.7 Hz, 2H), 7.13 (t, J = 9.0 Hz, 2H), 5.99 (s, 1H), 5.73 (s, 1H), 3.72 (t, J = 6.6 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H).

Compound 30 (1.2 g, mmol) was dissolved in Conc H2SO4 (4 mL) and heated at 60°C for 1 h. After cooling to rt, the mixture was diluted with EtOAc (200 mL) and washed with sat’d NaHCO3 (2×200 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated under vacuum followed by column chromatography on silica gel to afford 2-(2-chloro-ethyl)-5-fluoro-indan-1-one, 31 in quantitative yield. 1H NMR (CDCl3): δ 7.76 (dd, J = 8.4, 5.4 Hz, 1H), 7.05–7.14 (m, 2H), 3.80–3.86 (m, 1H), 3.68–3.78 (m, 1H), 3.42 (dd, J = 17.1, 7.8 Hz, 1H), 2.90–2.98 (m, 1H), 2.83 (dd, J = 17.1, 4.2 Hz, 1H), 2.38–2.49 (m, 1H), 1.86–1.98 (m, 1H).

4.1.2. 2′-(2-Chloroethyl)-5′-fluoro-2′,3′-dihydrospiro[[1,3]dioxolane-2,1′-indene], 33

A solution of 2-(2-chloro-ethyl)-5-fluoro-indan-1-one (5 g, 23.5 mmol), ethylene glycol (5 mL), TsOH (100 mg) in toluene (50 mL) was refluxed under N2 for 48 h. Water was removed by azeotropic distillation and the reaction was monitored by 1H NMR. The reaction was quenched by addition of Et3N (1 mL), diluted with EtOAc (250 mL), washed with sat NaHCO3, (25 mL), H2O (25 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated in under vacuum to dryness yielding a mixture of 2-(2-chloro-ethyl)-5-fluoro-indan-1-one and its ethylene acetal in a ratio of 1/4. 2-(2-chloroethyl)-5-fluoro-indan-1-one was removed by reducing to its 2-(2-chloro-ethyl)-5-fluoro-indan-1-ol with NaBH4 in MeOH, followed by column chromatography on silica gel which afforded 2′-(2-chloroethyl)-5′-fluoro-2′,3′-dihydrospiro[[1,3]dioxolane-2,1′-indene] 33 (4.5 g, 75%). 1H NMR (CDCl3): δ 7.24–7.28 (m, 1H), 6.89–6.96 (m, 2H), 4.22–4.28 (m, 1H), 4.07–4.16 (m, 3H), 3.70–3.74 (m, 1H), 3.62–3.68 (m, 1H), 3.55–3.61 (m, 1H), 3.07 (dd, J = 14.7, 6.6 Hz, 1H), 2.71–2.75 (m, 1H), 2.60–2.68 (m, 1H), 2.11–2.20 (m, 1H), 1.91–2.00 (m, 1H).

4.1.3. 2-(2-(4-(4-Chlorophenyl)piperazin-1-yl)ethyl)-5-fluoro-indan-1-one hydrochloride, 8

A mixture of 33 (1.2 g, 4.67 mmol), 1-(4-chlorophenyl)piperazine dihydrochloride (1.3 g, 5.6 mmol), KI (100 mg), K2CO3 (1.2 g, 8.75 mmol) in DME (10 mL) was heated to reflux under N2 for 12 h. The mixture was directly purified through column chromatography on silica gel to afford 2-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-5-fluoro-indan-1-one ethylene acetal. The product was dissolved in wet MeOH and TsOH was added with stirring at rt. After stirring at rt for 12 h, the solution was diluted with EtOAc (450 mL) and washed with saturated NaHCO3 (40 mL). The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated in vacuo to dryness and column chromatographed on silica gel to give 2-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-5-fluoro-indan-1-one, 8. The product was converted to the hydrochloride salt and further crystallization from MeOH-Et2O afforded the HCl salt (450 mg, yield 28%), mp 202–203 °C. 1H NMR (DMSO-d6): δ 11.15 (brs, 1H), 7.72 (dd, J = 8.7, 4.5 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.24–7.28 (m, 3H), 7.00 (d, J = 9.0 Hz, 2H), 3.77–3.80 (m, 2H), 3.53–3.56 (m, 2H), 3.28–3.36 (m, 2H), 3.12–3.22 (m, 4H), 2.81–2.90 (m, 2H), 2.22–2.78 (m, 1H), 1.84–1.96 (m, 2H). Calcd for C21H23Cl2FN2O: C 61.62, H 5.66, N 6.84; Found: C 61.38, H 5.58, N 6.77.

4.1.4. 2-(2-Chloroethyl)-indan-1-one

A mixture of 4-chloro-1-phenyl-butan-1-one (10 g, 54 mmol), hexanmethylene triamine (10.5 g, 75 mmol) in Ac2O (25 mL) was refluxed under N2 for 18 h. After cooling to rt, the mixture was diluted with CHCl3 (500 mL) and then washed with HCl solution (10%, 2×300 mL), H2O (300 mL), and saturated aq NaHCO3 (300 mL). The organic layer was dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel to afford 4-chloro-2-methylene-1-phenyl-butan-1-one (2.8 g, 26%). 1H NMR (CDCl3): δ 7.75–7.78 (m, 2H), 7.52–7.56 (m, 1H), 7.42–7.47 (m, 2H), 6.01 (s, 1H), 5.78 (s, 1H), 3.73 (t, J = 6.6 Hz, 2H), 2.95 (t, J = 6.6 Hz, 2H). The 4-chloro-2-methylene-1-phenyl-butan-1-one (1.2 g, 6.15 mmol) was dissolved in conc H2SO4 (4 mL) and heated at 60 °C for 1 h. The mixture was allowed to cool to rt, diluted with EtOAc (200 mL), washed with saturated aq NaHCO3 (2×200 mL) and dried over Na2SO4. The solution was filtered, solvent was removed in vacuo and the residue was purified by column chromatography on silica gel to afford 2-(2-chloro-ethyl)-indan-1-one in quantitative yield. 1H NMR (CDCl3): δ 7.75 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.45–7.48 (m, 1H,), 7.38 (t, J = 7.2 Hz, 1H), 3.75–3.86 (m, 1H), 3.68–3.74 (m, 1H), 3.39–3.47 (m, 1H), 2.89–2.94 (m, 1H), 2.80–2.88 (m, 1H), 2.38–2.47 (m, 1H), 1.88–1.96 (m, 1H).

4.1.5. 2-{2-[4-(4-Chloro-phenyl)-piperazin-1-yl]-ethyl}-indan-1-one tosylate, 9

A solution of 2-(2-chloroethyl)-indan-1-one (5 g, 25.6 mmol), ethylene glycol (5 mL), p-toluene sulfonic acid (TsOH, 100 mg) in toluene (50 mL) was refluxed under N2 for 48 h and water was removed by azeotropic distillation. The reaction was monitored by 1H NMR until a conversion of 80% was achieved. The reaction was quenched by the addition of Et3N (1 mL), diluted with EtOAc (250 mL), washed with saturated aq NaHCO3, (25 mL) and H2O (25 mL). The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated in vacuo to dryness to afford a mixture of 2-(2-chloro-ethyl)-indan-1-one and its ethylene acetal in a ratio of 1/4. 2-(2-Chloroethyl)-indan-1-one was removed by reducing to its 2-(2-chloro-ethyl)-indan-1-ol with NaBH4 in MeOH, followed by column chromatography on silica gel to afford 2-(2-chloro-ethyl)-indan-1-one ethylene acetal (4.4 g, 72%). 1H NMR (CDCl3):): 7.20–7,32 (m, 4H), 4.28–4.31 (m, 1H), 4.08–4.19 (m, 3H), 3.52–3.63 (m, 1H), 3.65–3.74 (m, 1H), 3.04–3.15 (m, 1H), 2.62–2.74 (m, 2H), 2.12–2.24 (m, 1H), 1.92–2.05 (m, 1H). A mixture of 2-(2-chloroethyl)indan-1-one ethylene acetal (1.0 g, 4.18 mmol), 1-(4-chlorophenyl)-piperazine dihydrochloride (1.4 g, 5.19 mmol), KI (100 mg), K2CO3 (1.2 g, 8.75 mmol) in DME (10 mL) was heated to reflux under N2 for 12 h. The mixture was allowed to cool to rt and then directly purified through column chromatography on silica gel to afford an oily residue. Without characterization, the product was dissolved in MeOH and p-toluene sulfonic acid (800 mg) was added with stirring at rt. After stirring for 12 h, the solution was diluted with EtOAc (450 mL) and washed with sat NaHCO3 (40 mL). The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated in vacuo to dryness. The resulting residue was purified by column chromatography on silica gel to afford 2-{2-[4-(4-chlorophenyl)-piperazin-1-yl]-ethyl}indan-1-one. The product was converted to p-toluenesulfonate and crystallized in MeOH-Et2O to afford the p-toluenesulfonate salt, 9 (610 mg, 28%). Mp 215–216 °C; 1H NMR (DMSO-d6): 9.57 (brs, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 3.80–3.85 (m, 2H), 3.58–3.62 (m, 2H), 3.25–3.39 (m, 2H), 3.11–3.21 (m, 2H), 2.92–3.00 (m, 2H), 2.80–2.87 (m, 1H), 2.72–2.77 (m, 1H), 2.24 (s, 3H), 2.16–2.22 (m, 1H), 1.84–1.93 (m, 1H). Calcd for C28H31ClN2O4S.0.8H2O: C 62.11, H 6.07, N 5.17; Found: C 62.09, H 6.06, N 5.08.

4.1.6. 2-(2-Chloroethyl)-5-fluoroindane, 32

Amalgamated zinc is prepared by stirring a mixture of zinc (1.2 g), HgCl2 (120 mg) in H2O (5 mL) with conc HCl (0.1 mL) at rt. After stirring for 5 min, the mixture was decanted, followed by the addition of H2O (1 mL), conc HCl (1.75 mL), toluene (10 mL), and 2-(2-chloroethyl)-5-fluoro-indan-1-one, 31 (2.0 g, 9.43 mmol). The mixture was refluxed with stirring for 12 h, allowed to cool to rt and the solid was filtered off. The collected filtrate was diluted with EtOAc (200 mL), separated and the organic layer was washed with H2O (50 mL) and saturated aq NaHCO3 (50 mL). The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated under vacuum. The resulting residue was purified by column chromatography on silica gel to afford 32 (1.68 g, 90%). 1H NMR (CDCl3): δ 7.09 (dd, J = 7.8, 4.8 Hz, 1H), 6.81–6.88 (m, 2H), 3.60 (t, J = 7.2 Hz, 2H), 3.00–3.08 (m, 2H), 2.68–2.73 (m, 1H), 2.54–2.63 (m, 2H), 1.94–2.02 (m, 2H).

4.1.7. 1-(4-Chlorophenyl)-4-(2-(5-fluoro-2,3-dihydro-1H-inden-2-yl)ethyl)piperazine dihydrochloride, 10

A mixture of 32 (0.8 g, 4.0 mmol), 1-(4-chlorophenyl)piperazine dihydrochloride (1 g, 4.3 mmol), KI (150 mg), K2CO3 (1.2 g, 8.7 mmol) in DME (10 mL) was heated to reflux under N2 for 12 h. The mixture was directly purified through column chromatography on silica gel to afford 1-(4-chloro-phenyl)-4-[2-(5-fluoro-indan-2-yl)-ethyl]-piperazine, 9. The product was converted to the hydrochloride salt immediately and then recrystallized from MeOH-Et2O (0.78 g, 45%), mp 194–196 °C. 1H NMR (DMSO-d6): 11.00 (brs, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.17 (dd, J = 5.7, 8.1 Hz, 1H), 6.99 (m, 3H), 6.90 (dt, J = 2.4, 8.4 Hz, 1H), 3.77 (d, J = 11.1 Hz, 2H), 3.53 (d, J = 11.1 Hz, 2H), 3.07–3.19 (m, 6H), 2.96–3.05 (m, 4H), 2.45–2.60 (m,1H), 1.88–1.96 (m, 2H). Calcd for C21H26Cl3FN2: C 58.41, H 6.07, N 6.49; Found: C 58.56, H 6.01, N 6.49.

4.1.8. 3-(4-Fluorophenoxy)propan-1-ol, 36a

A mixture of 4-fluorophenol (1.12g, 10 mmol), 3-chloropropanol (1.4 g, 15 mmol), KI (50 mg), K2CO3 (2.76 g, 20 mmol) in iPrOH was refluxed under N2 for 1h. The mixture was diluted with EtOAc (200 mL), washed with H2O (50 mL) and then brine (50 mL). The organic layer was dried with Na2SO4, and filtered. The filtrate was concentrated in vacuo, followed by distillation in vacuo to give the intermediate, 3-(4-fluorophenoxy)propan-1-ol, 36a (1.53 g, 90%). 1H NMR (CDCl3): δ 6.96 (t, J = 8.4 Hz, 2H), 6,84 (dd, J = 9.0, 4.5 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.84–3.87 (m, 2H), 1.99–2.07 (m, 2H).

4.1.9. 3-(4-Fluorophenoxy)propyl methanesulfonate, 37a

To a solution of 36a (1.3 g, 7.6 mmol), Et3N (3 mL) in CH2Cl2 (10 mL) was added at rt MsCl (0.8 mL, 10.3 mmol). The mixture was stirred at rt for 12 h, solvent was removed and the residue was purified through column chromatography on silica gel, to yield 3-(4-fluorophenoxy)propyl methanesulfonate, 37a (1.79 g, 95%). 1H NMR (CDCl3): δ 6.97 (t, J = 8.1 Hz, 2H), 6.83 (dd, J = 9.0, 4.5 Hz, 2H), 4.44 (t, J = 6.0 Hz, 2H), 4.05 (t, J = 6.0 Hz, 2H), 2.18–2.23 (m, 2H).

4.1.10. 3-((4-Fluorophenyl)thio)propan-1-ol, 36b

A mixture of 4-fluorobenzenthiol (1.55g, 12.1 mmol), 3-chloropropanol (2.26 g, 27.65 mmol), KI (100 mg), K2CO3 (3.3 g, 23.9 mmol) in iPrOH (10 mL) was refluxed under N2 for 1h. The mixture was diluted with EtOAc (200 mL), and washed with water (50 mL), brine (50 mL). The organic layer was dried with Na2SO4, and filtered. The filtrate was concentrated in vacuo, and followed by distillation in vacuo to give product 3-(4-fluorophenylthio)propan-1-ol, 36b (1.62 g, 72%). 1H NMR (CDCl3): δ 7.35 (dd, J = 8.4, 5.4 Hz, 2H), 6.99 (t, J = 8.4 Hz, 2H), 3.76 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2H), 1.80–1.89 (m, 2H).

4.1.11. 3-(4-Fluorophenylthio)propyl-4-methylbenzenesulfonate, 37b

To a solution of 3-(4-fluorophenylthio)-propan-1-ol (1 g, 5.4 mmol), Et3N (2 mL)in CH2Cl2 (10 mL) was added at rt TsCl (1.54 g, 8.1 mmol). The mixture was stirred at room temperature for 12 h, and then followed by directly purification through column chromatography on silica gel, and provided 3-(4-fluorophenylthio)propyl 4-methylbenzenesulfonate (1.72 g, 94%). 1H NMR (CDCl3): δ 7.77 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.30 (dd, J = 5.4, 8.4 Hz, 2H), 6.97 (d, J= 8.7 Hz, 2H), 4.13 (t, J = 8.0 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 1.85–1 92 (m, 2H).

4.1.12. 3-(4-Fluorophenylthio)propyl methanesulfonate, 37c

To a solution of 3-(4-fluorophenylthio)propan-1-ol (1.2 g, 6.45 mmol), Et3N (3 mL) in CH2Cl2 (10 mL) was added at rt MsCl (1 mL, 12.7 mmol). The mixture was stirred at room temperature for 12 h, and then followed by directly purification through column chromatography on silica gel, and provided 3-(4-fluorophenylthio)propyl methanesulfonate (1.60 g, 94%). 1H NMR (CDCl3): δ 7.37 (dd, J = 9.0, 4.8 Hz, 2H), 7.00 (t, J = 9.0 Hz, 2H), 4.30 (t, J = 8.0 Hz, 2H), 3.00 (s, 3H), 2.78 (t, J = 7.2 Hz, 2H), 1.99–2.04 (m, 2H).

4.1.13. General procedure of alkylation of arylpiperazines, 15a–c

A mixture of aryl piperazines (0.4 mmol, 1 eq.) and K2CO3 (4 mmol, 4 eq.) was stirred and refluxed for 20 min in CH3CN (10 mL). To the mixture, a solution of substituted tosylsulfonates/mesylates/chlorides (0.53 mmol, 1.3 eq.) in CH3CN (5 mL) was added drop wise. The reaction mixture was refluxed overnight, diluted with EtOAc (100 mL), filtered and washed with brine. The organic layer was collected, dried (Na2SO4), filtered, and the solvent was evaporated. The residue obtained was chromatographed on a silica gel column using hexane and EtOAc combinations as eluent. The final compounds were obtained in moderate yields, converted to HCl salts where necessary and re-crystallized using appropriate solvents.

4.1.14. 1-(3-(4-Fluorophenoxy)propyl)-4-(5-methylpyridin-2-yl)piperazine, 15a

Yield 30 %, mp 79.6 °C. 1H NMR (CDCl3): δ 8.02 (s, 1H), 7.32 (dd, J = 8.7, 2.4 Hz, 1H,), 6.99–6.93 (m, 2H), 6.85–6.81 (m, 2H), 6.59 (d, J = 8.7 Hz, 1H,), 4.00 (t, J = 6.6 Hz, 2H,), 3.52–3.48 (m, 4H), 2.61–2.55 (m, 6H), 2.19 (s, 3H), 2.05–1.96 (m, 2H). 13C NMR (CDCl3): δ 158.74, 158.03, 155.59, 155.07, 155.04, 147.67, 138.41, 122.45, 115.90, 115.60, 115.46, 115.35, 107.03, 66.75, 55.23, 53.04, 45.60, 26.65, 17.34. Calcd for C19H24FN3O: C 69.28, H 7.34, N 12.76; Foun: C 69.47, H 7.30, N 12.50.

4.1.15. 1-(3-((4-Fluorophenyl)thio)propyl)-4-(5-methylpyridin-2-yl)piperazine, 15b

Yield 62 %, mp 85.5 °C. 1H NMR (CDCl3): δ 8.00 (d, J = 2.4 Hz, 1H,), 7.37–7.29 (m, 3H), 7.01–6.96 (m, 2H), 6.58 (d, J = 8.7 Hz, 1H), 3.48–3.45 (m, 4H), 2.92 (t, J = 7.5 Hz, 2H), 2.54–2.46 (m, 6H), 2.18 (s, 3H), 1.86–1.77 (m, 2H). 13C NMR (CDCl3): δ 163.32, 160.06, 158.04, 147.65, 138.40, 132.20, 132.09, 122.41, 116.14, 115.85, 107.01, 105.00, 57.12, 53.01, 45.62, 32.86, 26.39, 17.34. Calcd for C19H24FN3S: C 66.05, H 7.00, N 12.16; Found: C 66.06, H 7.03, N 12.13.

4.1.16. 1-(4-(4-Fluorophenyl)butyl)-4-(5-methylpyridin-2-yl)piperazine, 15c

Yield 22 %, mp 74 °C 1H NMR (CDCl3) δ 8.01 (d, J = 2.7 Hz, 1H,), 7.29 (dd, J = 8.7, 2.7 Hz, 1H), 7.14–7.09 (m, 2H), 6.98–6.92 (m, 2H), 6.58 (d, J = 8.7 Hz, 1H), 3.50–3.46 (m, 4H), 2.63–2.52 (m, 6H), 2.42–2.37 (m, 2H), 2.18 (s, 3H), 1.68–1.50 (m, 4H). 13C NMR(CDCl3): δ 162.78, 159.56, 158.11, 147.67, 138.39, 137.97, 137.93, 129.72, 129.62, 122.37, 115.12, 114.84, 106.99, 58.59, 53.09, 45.65, 34.98, 29.51, 26.34, 17.35. Calcd for C20H26FN3: C 73.36, H 8.00, N 12.83; Found: C 73.79, H 8.28, N 12.47.

4.1.17. General Procedure for the synthesis of n-chloro-N-(2-hydroxy-phenyl)alkylamide, 40, (n=2–5)

For n=2, a solution of 2-aminophenol (1.0 g, 9.16 mmol), 3-chloropropionylchloride, (1.39 g, 11 mmol, 1.2 eq) and Et3N (1.12 g, 11 mmol, 1.2 eq) in EtOAc (25 mL) was heated to reflux for 6–10 h. After allowing to cool to rt, EtOAc (100 mL) was added and the solution was washed once with 10% HCl (100 mL). The aqueous layer was extracted with EtOAc (3×100 mL) and the combined organic layers was dried over anhydrous Na2SO4 and concentrated by rotary evaporation under reduced pressure. The pure product, 3-chloro-N-(2-hydroxyphenyl)propanamide, 40 was obtained as a colorless solid by column chromatography using EtOAc: hexane (3:7) as eluent.

4.1.18. General procedure for the synthesis of (n-chloroalkyl)benzoxazole, 41, (n = 2–5)

A mixture of 3-chloro-N-(2-hydroxyphenyl)-propanamide (1g, 5.01 mmole) and polyphosphoric acid (PPA, 3g) was heated with magnetic stirring at 130 °C for 3–4 h. The reaction mixture was poured into ice-water (50 mL), neutralized with saturated aq NH3, and extracted with EtOAc (2 × 50 mL). The combined extract was washed with H2O, brine, dried (Na2SO4), and concentrated to give the crude product which was purified by column chromatography using EtOAc: hexane (1:9) to give 2-(2-chloroethyl)benzoxazole as a pale yellow oily liquid.

4.1.19. General procedure for the synthesis of 2-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-benzoxazole

A mixture of 2-(2-chloroethyl)benzoxazole, (100 mg, 0.55 mmol), 4-chlorophenyl-piperazine, (108 mg, 0.55 mmol), and K2CO3 (433 mg, 3.30 mmol) in CH3CN (3 mL) was heated at reflux for 12–24 h. After cooling to rt, the solvent was removed under vacuum, H2O (10 mL) was added and the solution was extracted with EtOAc (3×50 mL). The pooled organic layer was washed with brine, dried over Na2SO4 and concentrated by rotary evaporation at reduced pressure. The residue was purified by column chromatography using EtOAc:hexane (9:1) as eluent to yield the pure product (2-{2-[4-(4-chlorophenyl)piperazin-1-yl]-ethyl}benzoxazole) as a colorless solid. The other benzoxazoles were similarly prepared.

4.1.20. 2-(5-(4-(4-Chlorophenyl)piperazin-1-yl)pentyl)benzo[d]oxazole, 19

Yield 45%, mp 110–112 °C 1H NMR (CDCl3): δ 7.68–7.65 (m, 1H), 7.50–7.46 (m, 1H), 7.31–7.28 (m, 2H), 7.19 (d, J = 9.3 Hz, 2H), 6.82 (d, J = 9.3 Hz, 2H), 3.17–3.13 (m, 4H), 2.95 (t, J = 7.5 Hz, 2H), 2.59–2.56 (m, 4H), 2.40 (t, J = 7.5 Hz, 2H), 1.98–1.91 (m, 2H), 1.66–1.58 (m, 4H). Calcd for C22H26ClN3O: C 68.83, H 6.83, N 10.95; Found: C 69.12, H 6.86, N 10.72.

4.1.21. 2-(4-(4-(4-Chlorophenyl)piperazin-1-yl)butyl)benzo[d]oxazole, 20

Yield 70%, mp 127–129 °C 1H NMR (CDCl3): δ 7.68–7.65 (m, 1H), 7.49–7.46 (m, 1H), 7.31–7.28 (m, 2H), 7.19 (d, J = 9.3 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 3.14 (t, J = 4.8 Hz, 4H), 2.98 (t, J = 7.5 Hz, 2H), 2.58 (t, J = 4.8 Hz, 4H), 2.45 (t, J = 7.5 Hz, 2H), 1.98–1.93 (m, 2H), 1.72–1.62 (m, 2H). Calcd for C21H24ClN3O 0.18 H2O: C 67.60, H 6.48, N 11.26; Found: C 67.69, H 6.61, N 10.98.

4.1.22. 2-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propyl)benzo[d]oxazole, 21

Yield 37%, mp 98–99 °C 1H NMR (CDCl3): δ 7.68–7.65 (m, 1H), 7.49–7.46 (m, 1H), 7.30–7.28 (m, 2H), 7.18 (d, J = 9.3 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 3.09 (t, J = 5.1 Hz, 4H), 3.01 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 5.1 Hz, 4H), 2.53 (t, J = 6.9 Hz, 2H), 2.14–2.09 (m, 2H). Calcd for C20H22ClN3O: C 67.50, H 6.23, N 11.81; Found: C 67.28, H 6.35, N 11.56.

4.1.23. 2-(2-(4-(4-Chlorophenyl)piperazin-1-yl)ethyl)benzo[d]oxazole, 22

Yield 58%, mp 110–112 °C 1H NMR (CDCl3): δ 7.69–7.66 (m, 1H), 7.52–7.47 (m, 1H), 7.32–7.30 (m, 2H), 7.19 (d, J = 9.3 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 3.18–3.14 (m, 6H), 3.01 (t, J = 6.9 Hz, 2H), 2.70 (t, J = 5.1 Hz, 4H). Cacld for C19H20ClN3O: C 66.76, H 5.90, N 12.29; Found C 67.06, N 5.97, H 11.90.

4.1.24. General Method for Tosylated alkyl benzofurans

The method reported in Bakunova et al,16 was followed to construct the benzofuran moiety. A mixture of 2- Iodophenol (3g, 1eq), alkyl-1-yn-1-ol (1.1eq), and copper (I) oxide (1.36 g, 0.7eq) in dry pyridine (15 mL) was stirred at 100–120 °C overnight. The mixture was allowed to cool to rt, diluted with EtOAc (50 mL), filtered through celite and concentrated. The residue was dissolved in EtOAc (100 mL), washed with 2 M HCl (50 mL) and brine (100 mL), dried over Na2SO4, and concentrated. The residue was, then, dissolved in CH2Cl2 (10 mL) and TsCl (1.1eq) and Et3N (1.4eq) were added at rt while stirring overnight. The solution was diluted with EtOAc (50 mL), washed with H2O (3 × 50 mL) and brine (50 mL). The pooled organic solvent was dried (Na2SO4), concentrated under reduced pressure and purified using column chromatography with hexane:EtOAc (7:3) as the eluent.

4.1.25. General Procedure for Benzofuran Coupling

To a stirred solution of CH3CN (10 mL) and Et3N (2 eq), the appropriate haloalkylbenzofuran (1eq) and an arylcycloalkylamine (1.1eq) were added and refluxed overnight. The solution was allowed to cool to rt, diluted with EtOAc (50 mL) and washed with H2O and brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified using column chromatography with hexane:EtOAc (6:4) as the eluent to afford an orange-yellow solid.

4.1.26. 1-(2-(Benzofuran-2-yl)ethyl)-4-(4-chlorophenyl)piperazine, 23

Yield 93%, mp 166.6 – 168.2 °C 1H NMR (CDCl3): δ 7.49 (dd, J = 4.5, 4.5 Hz, 1H), 7.42 (d, J = 12.0 Hz, 1H), 7.24 (m, 4H), 6.87 (d, J = 9.0 Hz, 2H), 6.46 (s, 1H), 3.19 (t, J = 15 Hz, 4H), 3.03 (t, J = 9.0 Hz, 2H), 3.85 (t, J = 9.0 Hz, 2H), 2.70 (t, J = 15 Hz, 4H). 13C NMR (CDCl3): δ 157.4, 154.3, 149.98, 128.98, 124.3, 123.2, 122.2, 120.2, 117.3, 110.07, 102.3, 56.1, 52.8, 49.3, 26.2. Calcd for C20H21ClN2O: C 70.48, H 6.21, N 8.22; Found: C 70.63, H 6.30, N 8.25.

4.1.27. 1-(3-(Benzofuran-2-yl)propyl)-4-(4-chlorophenyl)piperazine, 24

Yield 90%, mp 84.8 – 85.9 °C 1H NMR (CDCl3): δ 7.49 (dd, J = 6.0, 6.0 Hz, 1H), 7.41 (d, J = 12.0 Hz, 1H), 7.19 (m, 4H), 6.84 (d, J = 9.0 Hz, 2H), 6.41 (s, 1H), 3.15 (t, J = 15 Hz, 4H), 2.83 (t, J = 9.0 Hz, 2H), 2.62 (t, J = 12.0 Hz, 2H), 2.49 (t, J = 9 Hz, 4H), 1.97 (m, 2H). 13C NMR (CDCl3): δ 158.5, 154.5, 149.98, 128.94, 124.25, 123.21, 122.47, 120.24, 117.22, 110.75, 102.1, 57.66, 53.05, 49.11, 26.32, 24.88. Calcd for C21H23ClN2O·0.3H2O: C 70.01, H 6.43, N 7.78; Found: C 69.9, H 6.41, N 7.42.

4.1.28. 2-(3-(Benzofuran-2-yl)propyl)-5-(4-chlorophenyl)-2,5-diazabicyclo[2.2.1]heptane, 25

Yield 70%, mp 88.1 – 89.5 °C. 1H NMR (CDCl3): δ 7.46 (dd, J = 5.4, 4.5 Hz,1H), 7.19 (m, 1H), 7.17 (m, 1H), 7.13 (d, J = 9.6 Hz, 2H), 6.45 (d, J = 12.0 Hz, 2H), 6.35 (s, 1H), 4.18 (br s, 1H), 3.57 (br s, 1H), 3.35 (dd, J = 4.5, 3.3 Hz, 1H,), 3.22 (d, J = 9 Hz, 1H), 2.99 (dd, J = 4.5, 4.5 Hz, 1H), 2.78 (t, J = 9.0 Hz, 2H), 2.55 (m, 4H), 1.94 (t, J = 9 Hz, 2H), 1.84 (t, J = 9 Hz, 2H). 13C NMR (CDCl3): δ 158.9, 154.2, 145.7, 128.9, 123.3, 122.2, 120.9, 120.05, 113.8, 110.6, 102.1, 61.7, 57.7, 56.9, 52.05, 36.3, 27.1, 26.01. Calcd for C22H23ClN2O: C 72.02, H 6.32, N 7.64; Found: C 71.75, H 6.38, N 7.51.

4.1.29. 1-(3-(Benzofuran-2-yl)propyl)-4-(4-chlorophenyl)-1,4-diazepane dihydrochloride, 26

Yield 89%, mp 138.1 – 139.9 °C. 1H NMR (CDCl3): δ 7.46 (dd, J = 6.0, 4.8 Hz, 1H), 7.39 (dd, J = 6.0Hz, 3.3 Hz, 1H), 7.14–7.22 (m, 2H), 7.12 (d, J = 7.2 Hz, 2H), 6.57 (d, J = 6.0 Hz, 2H), 6.35 (s, 1H), 3.53 (t, J = 6.0 Hz, 2H), 3.44 (t, J = 9.0 Hz, 2H), 2.73–2.81 (m, 4H), 2.66 (t, J = 6.0 Hz, 2H), 2.58 (t, J = 6.0 Hz, 2H), 1.91–2.00 (m, 4H). Calcd for C22H27Cl3N2O·0.5H2O: C 63.77, H 6.32, N 6.76; Found: C 63.78, H 6.35, N 6.61.

4.1.30. 2-(3-Chloropropyl)benzo[d]thiazole, 48

A mixture of 2-aminobenzenethiol (10 g, 80 mmol) and 4-chlorobutyl chloride (14 g, 99 mmol) in toluene (100 mL) was stirred for 48 h at rt. The mixture was diluted with EtOAc (300 mL) and washed with saturated aq NaHCO3 (2 × 100 mL). The organic layer was dried with Na2SO4, and filtered. The filtrate was concentrated in vacuo, followed by chromatography on silica gel to afford 2-(3-chloropropyl)benzo[d]thiazole (13.5 g, 80%.) as an oil. 1H NMR (CDCl3): δ 7.98 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 8.4 Hz, 1H), 7.37 (t, J = 8.4 Hz, 1H), 3.69 (t, J = 6.6 Hz, 2H), 3.30 (t, J = 7.5 Hz, 2H), 2.34–2.42 (m, 2H).

4.1.31. 2-(3-(4-(Pyrimidin-2-yl)piperazin-1-yl)propyl)benzo[d]thiazole trihydrobromide, 28

A mixture of 2-(3-chloropropyl)benzothiazole (1.5 g, 7.08 mmol), 2-(piperazin-1-yl)pyrimidine dihydrochloride (1.6 g, 6.7 mmol), KI (200 mg), K2CO3 (1.2 g, 8.7 mmol) in DME (10 mL) was heated to reflux under N2 for 12 h. The mixture was diluted with EtOAc (400 ml) and washed with brine (50 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo to dryness and the residue was column chromatographed on silica gel to afford 2-[3-(4-pyrimidin-2-yl-piperazin-1-yl)propyl]benzothiazole, 28. The product was converted into the HBr salt, and recrystallized from MeOH-Et2O (865 mg, 21%), mp > 260 °C. 1H NMR (DMSO-d6): δ 12.08 (brs, 2H), 10.13 (brs, 1H), 8.45 (d, J = 4.5 Hz, 2H), 8.07 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.48 (dt, J = 1.5, 8.1 Hz, 1H), 7.40 (dt, J = 1.5, 8.1 Hz, 1H), 6.78 (t, J = 4.5 Hz, 1H), 4.68 (d, J = 14.1 Hz, 2H), 3.63 (d, J = 14.1 Hz, 2H), 3.35–3.44 (m, 2H), 3.20–3.31 (m, 4H), 3.04–3.14 (m, 2H), 2.25–2.35 (m, 2H). Calcd for C18H24Br3N5S: C 37.13, H 4.16, N 12.03; Found: C 37.22, H 4.11, N 12.01.

4.2. Receptor binding studies

Binding affinities (Ki, nM) reported in Tables 1–4 were conducted by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) unless otherwise stated. Details of the methods and the radioligands used for the binding assays at each receptor were previously reported.22

Acknowledgments

This work was supported by NIH grant # 5SC1GM088451-01-04 and a Title III Grant to Florida A&M University. The work was also supported in part by RCMI grant # 5 G12 MD007582-30 and the Pharmaceutical Research Center NIH/NCRR grant # 1C06-RR12512-01.

Footnotes

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References

  • 1.a) Seeman P, Van Tol HHM. Trends Pharmacol Sci. 1994;15:264. doi: 10.1016/0165-6147(94)90323-9. [DOI] [PubMed] [Google Scholar]; b) Van Tol HHM, Burnzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O. Nature. 1991;350:610. doi: 10.1038/350610a0. [DOI] [PubMed] [Google Scholar]; c) Beaulieu J-M, Gainetdinov RR. Pharmacol Rev. 2011;63:182. doi: 10.1124/pr.110.002642. [DOI] [PubMed] [Google Scholar]
  • 2.a) Bergauer M, Hübner H, Gmeiner P. Bioorg Med Chem Lett. 2002;12:1937. doi: 10.1016/s0960-894x(02)00316-5. [DOI] [PubMed] [Google Scholar]; b) McGough JJ. Pharmacogenomics. 2012;13:365. doi: 10.2217/pgs.12.5. [DOI] [PubMed] [Google Scholar]; c) Lynn DE, Lubke G, Yang M, McCracken JT, McGough JJ, Ishii J, Loo SK, Nelson SF, Smalley SL. Am J Psychiatry. 2005;162:906. doi: 10.1176/appi.ajp.162.5.906. [DOI] [PubMed] [Google Scholar]
  • 3.Ukai M, Mitsunaga H. Methods Find Exp Clin Pharmacol. 2005;27:645. doi: 10.1358/mf.2005.27.9.939341. [DOI] [PubMed] [Google Scholar]
  • 4.a) Yan Y, Mizuno T, Nitta A, Yamada K, Nabeshima T. Ann NY Acad Sci. 2004;1025:274. doi: 10.1196/annals.1316.034. [DOI] [PubMed] [Google Scholar]; b) Yan Y, Nitta A, Mizuno T, Nakajima A, Yamada K, Nabeshima T. Behav Brain Res. 2006;173:39. doi: 10.1016/j.bbr.2006.05.029. [DOI] [PubMed] [Google Scholar]
  • 5.Mamiya T, Matsumura T, Ukai M. Ann NY Acad Sci. 2004;1025:424. doi: 10.1196/annals.1316.052. [DOI] [PubMed] [Google Scholar]
  • 6.Yan Y, Pushparaj A, Le Strat Y, Gamaleddin I, Barnes C, Justinova Z, Goldberg SR, Le Foll B. Neuropsychopharmacology. 2012;37:685. doi: 10.1038/npp.2011.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.a) Sanna F, Corda MG, Melis MR, Piludu MA, Löber S, Hübner H, Gmeiner P, Argiolas A, Giorgi O. Pharmacol Biochem Behav. 2013;109:59. doi: 10.1016/j.pbb.2013.05.002. [DOI] [PubMed] [Google Scholar]; b) Patel MV, Kolasa T, Mortell K, Matulenko MA, Hakeem AA, Rohde JJ, Nelson SL, Cowart MD, Nakane M, Miller LN, Uchic ME, Terranova MA, El-Kouhen OF, Donnelly-Roberts DL, Namovic MT, Hollingsworth PR, Chang R, Martino BR, Wetter JM, Marsh KC, Martin R, Darbyshire JF, Gintant G, Hsieh GC, Moreland RB, Sullivan JP, Brioni JD, Stewart AO. J Med Chem. 2006;49:7450. doi: 10.1021/jm060662k. [DOI] [PubMed] [Google Scholar]; c) Kolasa T, Matulenko MA, Hakeem AA, Patel MV, Mortell K, Bhatia P, Henry R, Nakane M, Hsieh GC, Terranova MA, Uchic ME, Miller LN, Chang R, Donnelly-Roberts DL, Namovic MT, Hollingsworth PR, Martino B, El Kouhen O, Marsh KC, Wetter JM, Moreland RB, Brioni JD, Stewart AO. J Med Chem. 2006;49:5093. doi: 10.1021/jm060279f. [DOI] [PubMed] [Google Scholar]
  • 8.a) Galvez-Ruiz A, Arishi N. Saudi J Ophthalmol. 2013;27:241–6. doi: 10.1016/j.sjopt.2013.07.010. [DOI] [PMC free article] [PubMed] [Google Scholar]; b) Byoun H-S, Lee Y-J, Yi H-J. J Korean Neurosurg Soc. 2010;47:210–212. doi: 10.3340/jkns.2010.47.3.210. [DOI] [PMC free article] [PubMed] [Google Scholar]; c) Buxton N, Flannery T, Wild D, Bassi S. Br J Neurosurg. 2001;15:347–349. doi: 10.1080/02688690120072513. [DOI] [PubMed] [Google Scholar]; Egan RA, Pomeranz H. Neurology. 2002;59:293–294. doi: 10.1212/wnl.59.2.293-a. [DOI] [PubMed] [Google Scholar]; d) Habek M, Petravic D. Clin Neuropharmacol. 2006;29:165–167. doi: 10.1097/01.WNF.0000220815.86478.5F. [DOI] [PubMed] [Google Scholar]; e) Martí I, Martí Massó JF. Neurology. 2004;63:534. doi: 10.1212/01.wnl.0000130359.58099.8b. [DOI] [PubMed] [Google Scholar]; f) Monastero R, Pipia C, Camarda LK, Camarda R. J Neurol. 2001;248:141–142. doi: 10.1007/s004150170250. [DOI] [PubMed] [Google Scholar]
  • 9.Sood P, Idris NF, Cole S, Grayson B, Neill JC, Young AM. J Psychopharmacol. 2011;25:792–800. doi: 10.1177/0269881110387840. [DOI] [PubMed] [Google Scholar]
  • 10.a) Peprah K, Zhu XY, Eyunni SVK, Setola V, Roth BL, Ablordeppey SY. Bioorg Med Chem. 2012;20:1291. doi: 10.1016/j.bmc.2011.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]; b) Peprah K, Zhu XY, Eyunni SVK, Etukala JR, Setola V, Roth BL, Ablordeppey SY. Bioorg Med Chem. 2012;20:1671. doi: 10.1016/j.bmc.2012.01.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sikazwe DMN, Nkansah NT, Altundas R, Zhu XY, Roth BL, Ablordeppey SY. Bioorg Med Chem. 2009;17:1716. doi: 10.1016/j.bmc.2008.12.054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ablordeppey SY, Altundas R, Bricker B, Zhu XY, Eyunni VKSK, Jackson T, Khan A, Roth BL. Bioorg Med Chem. 2008;16:7291. doi: 10.1016/j.bmc.2008.06.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Vaidya T, Eisenberg R, Frontier AJ. ChemCatChem. 2011;3:1531. [Google Scholar]
  • 14.Yamamura S, Nishiyama S. Comp Org Syn. 1991;8:309. [Google Scholar]
  • 15.Zhu XY, Etukala JR, Eyunni SVK, Setola V, Roth BL, Ablordeppey SY. Eur J Med Chem. 2012;53:124. doi: 10.1016/j.ejmech.2012.03.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bakunova SM, Bakunov SA, Wenzler T, Barszcz T, Werbovetz KA, Brun R, Hall JE, Tidwell RR. J Med Chem. 2007;50:5807. doi: 10.1021/jm0708634. [DOI] [PubMed] [Google Scholar]
  • 17.http://www.cdc.gov/tobacco/data_statistics/fact_sheets/health_effects/effects_cig_smoking/ [accessed 2-10-2014]
  • 18.U.S. Department of Health and Human Services. A Report of the Surgeon General: How Tobacco Smoke Causes Disease: What It Means to You. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health; 2010. http://www.cdc.gov/tobacco/datastatistics/sgr/2010/consumerbooklet/pdfs/consumer.pdf[accessed 2-10-2014] [Google Scholar]
  • 19.a) Eisenberg MJ, Filion KB, Yavin D, Bélisle P, Mottillo S, Joseph L, Gervais A, O’Loughlin J, Paradis G, Rinfret S, Pilote L. CMAJ. 2008;179:135. doi: 10.1503/cmaj.070256. [DOI] [PMC free article] [PubMed] [Google Scholar]; b) Singh S, Loke YK, Spangler JG, Furberg CD. CMAJ. 2011;183:1359. doi: 10.1503/cmaj.110218. [DOI] [PMC free article] [PubMed] [Google Scholar]; c) Goldstein MG. J Clin Psychiatry. 1998;59:66. [PubMed] [Google Scholar]; d) Hays JT, Ebbert JO, Sood A. Mayo Clin Proc. 2009;84:730. doi: 10.4065/84.8.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Abdelfattah MAO, Lehman J, Abadi AH. Bioorg Med Chem Lett. 2013;23:5077. doi: 10.1016/j.bmcl.2013.07.033. [DOI] [PubMed] [Google Scholar]
  • 21.Kulagowski JJ, Broughton HB, Curtis NR, Mawer IM, Ridgill MP, Baker R, Emms F, Freedman SB, Marwood R, Patel S, Patel S, Ragan CI, Leeson PD. J Med Chem. 1996;39:1941. doi: 10.1021/jm9600712. [DOI] [PubMed] [Google Scholar]
  • 22.Shapiro DA, Renock S, Arrington E, Chiodo LA, Liu LX, Sibley DR, Roth BL, Mailman R. Neuropsychopharmacology. 2003;28:1400. doi: 10.1038/sj.npp.1300203. [DOI] [PubMed] [Google Scholar]

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