Article pubs.acs.org/jmc
Multiparameter Optimization in CNS Drug Discovery: Design of Pyrimido[4,5‑d]azepines as Potent 5‑Hydroxytryptamine 2C (5-HT2C) Receptor Agonists with Exquisite Functional Selectivity over 5‑HT2A and 5‑HT2B Receptors R. Ian Storer,*,†,# Paul E. Brennan,†,∞ Alan D. Brown,†,# Peter J. Bungay,§,# Kelly M. Conlon,‡ Matthew S. Corbett,∥ Robert P. DePianta,∥ Paul V. Fish,†,× Alexander Heifetz,⊥ Danny K. H. Ho,† Alan S. Jessiman,† Gordon McMurray,‡,# Cesar Augusto F. de Oliveira,∥ Lee R. Roberts,† James A. Root,‡ Veerabahu Shanmugasundaram,∥ Michael J. Shapiro,∥ Melanie Skerten,† Dominique Westbrook,‡ Simon Wheeler,† Gavin A. Whitlock,† and John Wright‡ †
Discovery Chemistry, ‡Discovery Biology, and §Pharmacokinetics, Dynamics and Metabolism, Sandwich Laboratories, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom ∥ Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States ⊥ Evotec (UK) Ltd., 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom S Supporting Information *
ABSTRACT: A series of 4-substituted pyrimido[4,5-d]azepines that are potent, selective 5-HT2C receptor partial agonists is described. A rational medicinal chemistry design strategy to deliver CNS penetration coupled with SAR-based optimization of selectivity and agonist potency provided compounds with the desired balance of preclinical properties. Lead compounds 17 (PF-4479745) and 18 (PF-4522654) displayed robust pharmacology in a preclinical canine model of stress urinary incontinence (SUI) and no measurable functional agonism at the key selectivity targets 5-HT2A and 5HT2B in relevant tissue-based assay systems. Utilizing recent advances in the structural biology of GPCRs, homology modeling has been carried out to rationalize binding and agonist efficacy of these compounds.
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INTRODUCTION
A recent industry-wide search for potent and selective 5HT2C agonists led to the discovery of lorcaserin (2) (APD-356) which gained FDA approval in 2012 for the treatment of obesity (Figure 1).4 At the time of carrying out the studies described herein, vabicaserin (3) (SCA-136) was also in clinical trials for the treatment of schizophrenia.5 In addition, multiple other small molecule 5-HT2C agonists have been reported to be
Sertonin, 5-hydroxytryptamine (5-HT) (1), is the endogenous agonist of at least 14 receptor subtypes, classified into seven families, 5-HT1−7. The 5-HT2 class has three members 2A, 2B, and 2C. Although 5-HT2A and 5-HT2B receptors are known to also be expressed peripherally, the expression of 5-HT2C receptors is believed to be restricted to the central nervous system (CNS). The 5-HT2C receptor has been viewed as an attractive drug target for many years with potential application for the treatment of a number of medical conditions including obesity, psychiatric disorders, sexual dysfunction, and urinary incontinence.1 More recently, selectivity over agonism of 5HT2A and 5-HT2B receptor subtypes has become a major imperative; 5-HT2A agonists are known to cause hallucinations and drive adverse cardiovascular (CV) effects,2 while 5-HT2B agonists have been associated with chronic irreversible cardiac valvulopathy and pulmonary hypertension, as illustrated by the market withdrawal of the unselective serotonergic agonist FenPhen in 1997.3 © XXXX American Chemical Society
Figure 1. Selected 5-HT2C agonists. Received: March 1, 2014
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and amines (5−11) led to improvements in both potency at 5HT2C and selectivity over both 5-HT2A and 5-HT2B (Table 1). Interestingly, these selectivity improvements were also broadly coupled with an unexpected decrease in Emax of agonism efficacy at 5-HT2C. Additionally, compounds containing aliphatic 4-amino substituents (8−11), were generally subject to P-glycoprotein (P-gp) mediated efflux as measured using an in vitro MDCK cell line transfected with P-gp.11 As efflux by Pgp at the blood−brain barrier has been well characterized as a mechanism that restricts entry of drugs into the CNS, it was recognized that in vivo functional efficacy of compounds acting as P-gp substrates may be limited by impairment of CNS penetration.12 The in vitro MDCK-MDR1 cultured cell monolayer assay has been demonstrated to be able to distinguish P-gp substrates from non-P-gp substrates by measuring efflux ratio (ER), whereby compounds with ER > 2.5 are considered to be significant substrates of P-gp.11,13 Compound 7 (R = MeO, non-P-gp substrate with low Emax of 22%) and compound 8 (R = MeHN, P-gp substrate with moderate Emax of 40%) exhibited the requisite levels of subtype selectivity of >100-fold over 5-HT2B and presented different levels of agonist Emax. As a result, both 7 and 8 were tested in an established human efficacy correlated preclinical in vivo canine model of stress urinary incontinence (SUI). This peak urethral pressure (PUP) model was used to measure dose-dependent increases in urethral tone relative to prototype lead compound 4 (Figure 3a).10 Refer to the Supporting Information for a detailed description of the PUP model and its translation to human disease. This dog model was used to inform on in vivo efficacy for the 5-HT2C mechanism and as a translatable efficacy surrogate for a range of diseases including SUI, obesity, erectile dysfunction, and psychotherapeutic disorders.6h Compound 7 did not exhibit increases in PUP to a degree viewed as biologically meaningful in comparison to PUP increases elicited at clinically relevant exposures of duloxetine and reboxetine, despite being freely CNS penetrant, as assessed by measuring unbound concentration in plasma and cerebrospinal fluid (CSF/free plasma ratio of 0.6, suggesting
in early clinical development or undergoing preclinical evaluation.6 Previously, Pfizer has disclosed several selective 5-HT2C receptor agonists, 7 including compounds containing a pyrimido[4,5-d]azepine template that led to the selection of compound 4 as a first generation lead from this series (Figure 2).8 Although compound 4 delivered favorable levels of efficacy
Figure 2. Compound 4 showed measurable 5-HT2B agonism.8a
and safety in preclinical studies, a weak signal for 5-HT2B agonism was observed in in vitro human colon tissue studies at high concentration.9 Considering potential safety risk implications of chronic dosing of a compound with even weak 5-HT2B agonism, further medicinal chemistry effort was focused toward design of a CNS penetrant 5-HT2C agonist candidate molecule with improved selectivity over 5-HT2B. At the same time it was necessary to retain good preclinical in vivo efficacy determined using a canine model of stress urinary incontinence (SUI) for clinical dose-prediction.10 This article describes the compound design rationale and results of these studies.
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RESULTS AND DISCUSSION Designing Increased Subtype Selectivity: 4-Substituted Pyrimidines. Initial work focused on the incorporation of small heteroatom-containing groups at the synthetically enabled 4-position of the pyrimidine to target directional polar electrostatic interactions. On the basis of previous knowledge of structure−activity relationships (SARs) for related templates, it was postulated that such modifications would retain agonist activity at 5-HT2C and lead to enhanced receptor subtype selectivity. In accordance with the hypothesis, the introduction of small heteroatom linked substituents such as alkoxyethers Table 1. 4-Substituted Pyrimidine Derivatives
compd
R
log D
5-HT2C EC50, nM (Emax)a,b
5-HT2C binding Ki, nM
5-HT2B EC50, nM (Emax)a,c
5-HT2A EC50, nM (Emax)a,d
MDCK-MDR1 ERe
>10000 (41%), n = 14
3400 (51%), n = 12
1.2
NTf
NTf
3.0
NTf
NTf
NTf
1.0
190 {164, 224} (75%), n = 19 160 {135, 209}, n = 19 1460 {1204, 1685} (12%), NTf n=4 >10000 (20%), n = 4 958 {458, 6483}, n=2 51 {29, 68} (22%), n = 6 36 {24, 53}, n = 5
>10000 (10000 (10000 (10000 (10000 (20% in the dog correlates with clinically relevant exposures and efficacy in humans for both compounds.10,14 (b) Recombinant, stably expressed human 5-HT2C CHO K1 cell line, FLIPR agonist dose−response curves illustrating differences in efficacy Emax.
sufficient CNS exposure). The lack of relative effect was likely due to low functional agonism (Emax = 22%, Figure 3b), rendering this compound a weak partial 5-HT2C agonist in vivo; it is plausible that this compound could act as a functional antagonist in the presence of a full agonist in vivo, although this has not been investigated. Compound 8 exhibited greater agonist efficacy (Emax = 40%, Figure 3b) and was able to elicit an increase in PUP but only at high plasma concentrations and also exhibited appreciable variability between animals, with some individuals not responding to compound treatment. Compound 8 was also a P-gp substrate (ER = 10) and likely to have restricted CNS penetration (CSF/free plasma ratio of 0.1), requiring higher plasma concentrations to achieve sufficient occupancy of 5-HT2C receptors in the CNS. Although these investigations halted the progression of both compounds 7 and 8, they provided key information to guide future design objectives by defining an acceptable target efficacy Emax of >40% in the FLIPR assay and confirmed the need for ER of 95% purity. LRMS (ESI, APCI) m/z 405 [M + H]+.
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Article
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0)1304 641854. E-mail: ian.storer@pfizer.com. Present Addresses #
R.I.S., A.D.B., P.J.B., G.M.: Pfizer Neusentis, The Portway Building, Granta Park, Cambridge, CB21 6GS, United Kingdom. ∞ P.E.B.: Structural Genomics Consortium, Target Discovery Institute, Nuffield Department of Medicine, Oxford, OX3 7BN, United Kingdom. × P.V.F.: UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, United Kingdom. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the Primary Pharmacology Group for screening data, Hau Gao for the cMDR efflux model, and support from Laure Hitzel and Juliette Love in the Separation and Analytical Services group for chiral analysis and preparative chromatography. We also thank Jianmin Sun for coordinating the collection of HRMS data on selected compounds.
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ABBREVIATIONS USED ADME, absorption, distribution, metabolism, and excretion; Boc, tert-butyloxycarbonyl; BBB, blood−brain barrier; Ceff, efficacious concentration; CHO, Chinese hamster ovary; CL, clearance; CSF, cerebrospinal fluid; CNS, central nervous system; CV, cardiovascular; CYP, cytochrome P450; DCM, dichloromethane; DLM, dog liver microsome; Dof, dofetilide; DOI, 2,5-dimethoxy-4-iodoamphetamine; dppb, dog plasma protein binding; ER, efflux ratio; FDA, Federal Drug Agency; FLIPR, fluorescence imaging plate reader; hep, hepatocytes; hERG, human ether-a-go-go-related gene; hppb, human plasma protein binding; HLM, human liver microsome; log D, partition coefficient between octanol and water at pH 7.4; MDCK, Madin−Darby canine kidney; MDR1, multidrug resistance gene 1; PAMPA, parallel artificial membrane permeability assay; PDB, Protein Data Bank; P-gp, Pglycoprotein; ppb, plasma protein binding; PUP, peak urethral pressure; RLM, rat liver microsome; SAR, structure−activity relationship; SDM, site directed mutagenesis; SUI, stress urinary incontinence; Tf, triflate (trifluoromethanesulfonate); TM, transmembrane; TPSA, topological polar surface area; VCD, vibrational circular dichroism
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Synthetic procedures, characterization, and purity analysis of all compounds; selected spectra for key compounds; details of the VCD determination of stereochemical assignment of compounds 16 and 17; detailed descriptions of computational modeling; procedures for the functional, binding, and tissue based assays; detailed in vivo methodology. This material is available free of charge via the Internet at http://pubs.acs.org. J
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dx.doi.org/10.1021/jm5003292 | J. Med. Chem. XXXX, XXX, XXX−XXX
