Accepted Article

Received Date : 22-Nov-2013 Accepted Date : 06-Jan-2014 Article type : Research Letter Research Letter Title

In Search of Potent 5–HT6 Receptor Inverse Agonists

Greg Hostetler, Derek Dunn, Beth Ann McKenna, Karla Kopec and Sankar Chatterjee* Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380-4245, USA *Corresponding author: Sankar Chatterjee, [email protected] Keywords. 5-HT6 receptor, CNS disorders, benzazepine.

Abstract. A series of non-sulfonamide/non-sulfone derived potent 5–HT6 receptor inverse agonists has been disclosed. Representative compound 9 (Ki = 14 nM) displayed selectivity against a set of family members as well as brain permeability 6h post oral administration. In addition, the separated enantiomers of compound 9 displayed difference in activity indicating the influence of chirality on potency.

5-hydroxytryptamine 6 (5–HT6) receptor is a prominent member of the serotonin receptor family (1). This receptor has been implicated to play a role in cognitive function in Alzheimer’s disease and schizophrenia, anxiety, obesity, depression and sleep-wake activity (2 – 5). Thus the receptor has become a recent target for the pharmaceutical intervention (6 - 8). In our effort to identify a synthetic inverse agonist / antagonist of this receptor, our internal chemical library was profiled on a high throughput screening (HTS) platform. From this engagement, the team encountered a 1-thia-4,7-diaza-spiro[4.4]nonane-3,6-dione-derived “hit” compound 1a [Ki of 5.73 µM against human 5-HT6 (h5-HT6) receptor, Fig. 1] (9). Subsequently, compound 1a acted as a launching pad for additional exploration of the series.

While a research program was aimed at developing the SAR around the central [5,5] – spiro motif (rings B/C) (9), a parallel program was initiated to explore whether the motif itself was needed for the potency of this class of compounds. Accordingly, the spiro bicyclic system in compound 1a was deconstructed into a linear array as exemplified by the generic structure E (Fig. 1). In addition, it was anticipated that if potent, the series would be notable for the absence of any sulfonamide or sulfone moiety in the framework, a frequent feature of literature reported This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12279 This article is protected by copyright. All rights reserved.

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potent inverse agonists/antagonists from various laboratories at the time (7). In this Letter, we disclose some preliminary results from our ongoing exploration from this parallel endeavor. Following two schemes were employed to generate various target compounds of interest. In Scheme 1, commercially available tboc–protected benzazepine amine 2 was coupled with substituted α-chloroethylphenyl derivatives 3 under microwave condition to generate adducts of generic structure 4 that were subsequently deprotected to produce the generic amine– hydrochloride salts 5. In Scheme 2, compound 2 was coupled with substituted acetophenones 6 to generate intermediate imines 7 that were reduced to generate the generic amines 4 followed by deprotection of the t-boc group to produce the generic targets 5.

Following a reported procedure, binding properties of the target compounds were assessed against recombinant human 5-HT6 (h5-HT6) receptors by displacement of [3H] LSD (a potent ligand of the receptor) and reported as Ki values from an average of two experiments, done in triplicate (10). Biologic data are shown in Table 1. Identification of compound 1a as a modestly active “hit” molecule encouraged us to profile additional available derivatives from the same class. This offered a more potent analog 1b (Fig. 1 and Table 1, Ki of 88 nM). In our seminal manuscript in this area, we described in detail how combination of these two results led us to introduce an azepine moiety (ring F, Table 1) in the western hemisphere of the designed targets in the first generation of targets as well as in the current series (9). As shown in Table 1, the initial analog 8 from this new series was ca. 80-fold more potent than the HTS-hit compound 1a. This provided support to the hypothesis that the combination of a central linear moiety in conjunction with an azepine system in the western region of the molecule could replace the architecturally rigid [5,5]-spiro bicyclic system of compound 1a. The enhancement in potency also revealed the need for the basic nitrogen atom in the western region. Introduction of an ortho-chloro group in the phenyl ring (ring D) generated compound 9 that was ca. 409-fold more potent than compound 1a indicating additional lipophilicity/steric bulk from this site might be beneficial to the potency. Moving the chlorine atom from ortho– to meta– position around the ring, potency was maintained (cf. compound 9 vs. compound 10) while moving it to para-position resulted in ca. two–fold loss in potency (cf. compound 11 vs. compound 9). Substitution of chlorine atom at position–2 of this phenyl moiety (ring C) with other members of the halogen family (compound 12 and compound 13, respectively) was not detrimental to potency. Ortho position also tolerated a methyl group (compound 14), a trifluoromethyl group (compound 15) as well as alkoxy groups (compounds 16 and 17). On the other hand, introduction of a phenyl moiety to the same position (compound 18) was detrimental indicating the requirement of an optimum balance between lipophilicity and steric limit from this site on potency. On the other hand, 2, 3–disubstitution of the phenyl ring with halogens was tolerated, di–chloro being preferred over di–fluoro (cf. compound 20 vs. compound 19). In expanding the scope of the SAR, the substitution of the hydrogen atom with a methyl group on azepine–N (ring F) was tolerated (cf. compound 21 vs. compound 9). Substitution of the methyl group at the chiral center with an ethyl group reduced the potency ca.

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four–fold (cf. compound 22 vs. compound 10) revealing the steric limit on potency from this site. Replacement of the hydrogen of the -NH group attached to ring B with a methyl group maintained the potency (cf. compound 23 vs. compound 9) suggesting this site tolerates some steric flexibility towards potency. Finally, compound 24 containing an acetyl group on azepine– N (ring F) displayed a reduction in potency re-confirming the requirement of the basic nitrogen on potency at this site. From the above series of target molecules, the representative compound 9 was further profiled against the various members of the serotonin family displaying >100–fold selectivity against each of following subtype: 5–HT2b, 5–HT2C, 5–HT3, 5–HT5a and 5–HT7 respectively, and >28–fold selectivity against 5HT1a. In rat pharmacokinetic (PK) studies, compound 9 was orally bioavailable and detected in the brain even after 6 h post oral administration. Finally, in order to probe the effect of the stereo center on the binding activity, compound 9 was separated into two enantiomers (optical purity >98% ee) via chiral column chromatography. They displayed >5– fold difference in binding potency (Ki of 7 nM vs. 38 nM, respectively); the determination of absolute configuration of either enantiomer remains an active area of research as well as additional profiling of the isomers. In conclusion in this Letter, we disclosed a series of non-sulfonamide/non-sulfone derived potent 5–HT6 receptor inverse agonists. From this study, representative compound 9 underwent further exploration displaying selectivity against a set of family members as well as brain permeability 6h post oral administration. In addition, the separated enantiomers of compound 9 displayed difference in potency suggesting a role played by the configuration of the chiral center on activity. Additional research is continuing. Acknowledgement Authors wish to acknowledge Drs. Edward R. Bacon and John P. Mallamo for their support and encouragement.

Conflict of interest Authors, as the employees of Cephalon, Inc. were engaged in discovering potent 5–HT6 antagonists. References 1. Vitalis, T., Ansorge. M. S., Dayer, A. G. (2013) Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes. Front Cell Neurosci; 7: 93 (doi: 10.3389/fncel.2013.00093). 2. Ramirez, M. J. (2013) 5-HT6 receptors and Alzheimer's disease. Alzheimer’s Res Ther; 5:15. (epub ahead of print). 3. Shimizu, S., Mizuguchi, Y., Ohno Y. (2013) Improving the Treatment of Schizophrenia: Role of 5-HT Receptors in Modulating Cognitive and Extrapyramidal Motor Functions. CNS Neurol Disord Drug Targets; Jul 10. [epub ahead of print]. 4. Wesołowska, A. (2010) Potential role of the 5-HT6 receptor in depression and anxiety: an overview of clinical data. Pharmacol Rep; 62:564–77.

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5. Ly, S., Pishadari, B., Lok, L. L., Hajos, M., Kocsis, B. (2013) Activation of 5-HT6 receptors modulates sleep-wake activity and hippocampal theta oscillation. ACS Chem Neurosci;4:191–9. 6. Marazziti, D., Baroni, S., Borsini, F., Picchetti, M., Falaschi, V., Catena-Dell, M. O. (2013) Serotonin receptors of type 6 (5-HT6): from neuroscience to clinical pharmacology. Curr Med Chem; 20: 371-7. 7. Rossé, G., Schaffhauser, H. (2010) 5-HT6 receptor antagonists as potential therapeutics for cognitive impairment. Curr Top Med Chem; 10:207–21. 8. Maher–Edwards, G., Zvartau–Hind, J. Hunter, A. J., Gold, M., Hopton, G., Jacobs, G., Davy, M., Williams, P. (2010) Double-blind, controlled phase II study of a 5-HT6 receptor antagonist, SB-742457, in Alzheimer's disease. Curr Alzheimer Res; 7:374–85. 9. Hostetler, G., Dunn, D., McKenna, B. A., Kopec, K., Chatterjee, S. (2013) 1-Thia-4,7-diazaspiro[4.4]nonane-3,6-dione: A Structural Motif for 5-hydroxytryptamine 6 Receptor Antagonism. Chem. Bio. Drug Design; (DOI: 10.1111/cbdd.12240). 10. Bacon, E. R., Bailey, T. R., Dunn, D. D., Hostetler, G. A., McHugh, R. J., Morton, G. C., Rossé, G. C., Salvino, J. M., Sundar, B. G., Tripathy, R. (2011) Patent application WO 2011/087712:1–222.(a)

Note a. Human 5-HT6 Receptor Binding Assay

Membrane preparation: Membranes were prepared from CHO-K1 cells stably transfected with the human 5-HT6 receptor (Euroscreen; ES-316-C). The cells were grown in Gibco Advanced DMEM-F12 (Cat # 12634010) containing 2% dialyzed FBS (Hyclone Cat # SH30079.03). The cells were harvested in phosphate buffered saline (PBS) containing 0.1 mM EDTA and pelleted by centrifugation (1000 x g), the supernatant was discarded and the pellets were stored at -80 oC prior to membrane preparation. Membranes were then prepared as follows. Briefly, frozen cell pellet was resuspended in a lysis buffer containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA and 1 complete EDTA-free protease inhibition tablet (Roche Applied Science, Indianapolis, IN) per 50 mL buffer and homogenized with a tissue homogenizer. The cell lysate was then centrifuged at 40,000 x g for 30 min at 4 oC to collect the membranes. The membrane pellets were washed in membrane buffer (50 mM Tris-HCl (pH 7.5), 0.6 mM EDTA, 5 mM MgCl2, 1 complete EDTAfree protease inhibitor tablet per 50 mL buffer) using a tissue homogenizer. The membranes were centrifuged at 40,000 x g for 30 min at 4 oC and the pellets were resuspended in membrane buffer containing 250 mM sucrose, and protein concentration was determined using the Comassie Plus kit (Pierce Biotechnology, Rockford, IL).

Receptor binding assay: Membranes prepared from cells expressing recombinant human 5-HT6 receptor (h5-HT6) were resuspended in assay buffer containing 50 mM Tris HCl (pH 7.4), 4 mM CaCl2, 10 μg/mL saponin and 0.1% (w/v) ascorbic acid. Membranes were preincubated using 1.75 μg membrane protein and 0.25 mg FlashBlue scintillation beads (Perkin Elmer catalogue # FB001) per well at 4 oC for 30 min. Vehicle or test compound and 4 nM [3H]LSD (PerkinElmer catalogue #

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NET638) were added and incubated for 3 hours at room temperature in a final volume of 80 μL in a 96-well plate. Test compounds or assay controls for total and non-specific binding were diluted in DMSO as 100x solutions and serially diluted by half log concentrations on a Perkin Elmer JANUS Automated Workstation. Serotonin (10 μM final concentration) was used to determine non-specific binding in the assay. Plates were read using the Microbeta Trilux 1450 LSC and luminescence counter. Data were analyzed by nonlinear regression using the doseresponse equation (variable slope) to calculate IC50 in XLfit4 (ID Business Solutions Inc.): y = (Bottom+((Top-Bottom)/(1+((IC50/x)^Hill slope)))) Binding of [3H]LSD to the h5-HT6 membranes was saturable with Bmax = 6.2 pmol/mg protein and Kd = 2.3 nM. Ki value was then calculated according to Cheng-Prussof method using the equation: Ki app = IC50 / (1+([radioligand]Kd)). Legends for Figure and Schemes Figure Figure 1. Chemical structures of initial lead 1 and current series E.

Schemes Scheme 1. Reagents and conditions: (a) catalytic NaI, diisopropylethyl amine, dioxane, heat at 130 oC in a microwave, 45 – 50 min, 50 – 60%; (b) 4 N HCl in dioxane, room temperature, 2 h, 80-90%.

Scheme 2. Reagents and conditions: (a) Toluene, reflux under Dean-Stark condition or in presence of 4Å molecular sieves; 5 – 6 h; (b) NaBH4, MeOH, 0 oC to room temp., 40 – 50% over two steps; (c) 4 N HCl in dioxane, room temperature, 2 h, 80-90%.

Title for Table Table 1. Biologic data for compounds 1a – b and 8 - 24a, b

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Table Table 1. Biological data for compounds 1a – b and 8 - 24a, b

X N

F

W

A N Y

2 3

1

D

6

R 4

5

Compound 1a 1b 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

X

H H H H H H H H H H H H H Me H H COMe

Y

H H H H H H H H H H H H H H H Me H

W

Me Me Me Me Me Me Me Me Me Me Me Me Me Me Et Me Me

R

Ki nMc

H 2-Cl 3-Cl 4-Cl 2-F 2-Br 2-Me 2-CF3 2-OMe 2-OCF3 2-Ph 2,3-F2 2,3-Cl2 2-Cl 3-Cl 2-Cl 2-Cl

5,730 88 72 14 10 31 21 11 34 36 27 14 236 39 10 8 39 19 1,200

a

Enzyme assay against recombinant h5-HT6; internal ligand [3H]LSD (binding of this ligand to the h5-HT6 membranes was saturable with Bmax = 6.2 pmol/mg protein and Kd = 2.3 nM). Ki value of a test compound was then calculated according to the Cheng-Prusoff method. b All compounds were tested as racemates. c Average of three experiments.

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Figure and Schemes Figure

Figure 1

R 3 4 5 R

A

D

2 O 1 N B S C O N H

Y

A N

X

D

H

1a R = H 1b R = OMe

E

Schemes Scheme 1 Cl tBoc

N

+

R

NH2 3

2 a

Me

X N N H 4 X = tBoc b 5 X= H . HCl

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R

z

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Scheme 2

O a

2 + Me

R

tBoc

Me

N N

R

7

6

b

4) X = tBoc c

Me

X N

5) X= H . HCl

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N H R

In search of potent 5-HT6 receptor inverse agonists.

A series of non-sulfonamide/non-sulfone derived potent 5-HT6 receptor inverse agonists has been disclosed. Representative compound 9 (Ki  = 14 nm) dis...
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