Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

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Development of spiroguanidine-derived a7 neuronal nicotinic receptor partial agonists Matthew D. Hill ⇑, Haiquan Fang, Sivarao V. Digavalli, Francine L. Healy, Lizbeth Gallagher, Debra Post-Munson, Ping Chen, Joanne Natale, Yulia Benitex, Daniel Morgan, Nicholas Lodge, Linda Bristow, John E. Macor, Richard E. Olson Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492-7660, USA

a r t i c l e

i n f o

Article history: Received 20 November 2016 Revised 3 December 2016 Accepted 3 December 2016 Available online xxxx Keywords: a7 nicotinic acetylcholine receptor Schizophrenia 5-HT3A receptor Spiroguanidine Immediate early genes

a b s t r a c t We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as a7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective a7 nAChR partial agonist, N-(6-methyl-1,3-benzoxazol-2-yl)-30 ,50 -dihydro-4azaspiro[bicyclo[2.2.2]octane-2,40 -imidazole]-20 -amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of a7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the a7 nACh receptor over the 5-HT3A receptor in this series suggested a significant difference in steric requirements between the two receptors. Ó 2016 Elsevier Ltd. All rights reserved.

Schizophrenia is a complex and debilitating psychiatric disorder that places a significant burden on society. Related healthcare expenses within the US market are estimated to be in the tens of billions of dollars to treat the disease. Beyond any financial impact, schizophrenia increases the rate of all-cause mortality (2–3-fold) and leads to a 10-year reduction in average life span.1 Positive symptoms can be effectively treated using existing therapies, but negative and cognitive symptoms remain challenging. Additionally, patient compliance with current standard of care remains difficult due to extrapyramidal motor effects and significant tolerability issues.1b Recent clinical data suggest that a7 neuronal nicotinic acetylcholine receptor (nAChR) agonism may provide an effective treatment for both cognitive and negative symptoms in patients with schizophrenia.2 The a7 nAChR is a ligand-gated ion channel that is centralized in regions of the brain that are associated with learning and memory.3 Knockout animal and other in vivo studies have been used to gauge the feasibility of a7 nAChR agonism to improve learning and memory.3,4 These studies indicate enhancement in animal learning and memory function, reversal of memory and sensory gating deficits, and anxiolytic properties can be achieved through augmentation of a7 nAChR activity,5 making the a7

⇑ Corresponding author. E-mail address: [email protected] (M.D. Hill).

nAChR an attractive target for new therapies to treat schizophrenia.6 Full agonists (Fig. 1) have been shown to rapidly desensitize the a7 nAChR, an effect that may impact in vivo efficacy.7 Therefore, our efforts were focused on partial agonists, which we expected to provide a lower potential for ion channel desensitization. We previously described potent carbamimidate-containing a7 nAChR partial agonists (e.g., BMS-902483) that demonstrated in vivo efficacy in cognition models.8 However, identification of a molecule with an acceptable off-target profile proved difficult; many early leads demonstrated low micromolar inhibition of the hERG potassium channel.9 Moreover, design of analogs selective for the a7 nAChR over serotonergic 5-HT3A receptor proved challenging due to high sequence homology between the two receptors.10 Herein we report a novel series of quinuclidine-containing spiroguanidine a7 nAChR partial agonists 1 designed to overcome these limitations (Scheme 1). Importantly, the spiroguanidine moiety provided a new vector (R, Fig. 1) that could potentially impact both a7 nAChR and 5-HT3A activities and hERG inhibition.11 The functionalized quinuclidine moiety is the most commonly described bioisostere of the quaternized ammonium functionality in acetylcholine, as exemplified by AR-R17779, one of the first selective a7 nAChR agonists identified (Fig. 1).12 Based on earlier SAR from the structurally similar carbamimidate series,8 this region of our spiroguanidine chemotype was held constant. The highly polar spiroguanidine region of this series (1) may act as a

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M.D. Hill et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

H

O O

N Me

N

N

nicotine

R

NH

N

O

O N

N

N

R

S NH

N

N

Het R

3 or

NH2 MeS

2

N SMe

N

N Het

H N Het

a

S Het

NCS

a

NH

N

N

5

Het

R

c

SMe

N

4

N

H N

H N

Het

S

b

H N Het N

1

Het

NH2

a

Het

SMe

N

SMe

1

4

hydrogen-bond acceptor, mimicking the acetate carbonyl in acetylcholine, and linking the lipophilic heteroaryl rings (Het) with the bridged bicyclic quinuclidine, two critical functions for target binding. The heteroaryl ring proved crucial for a7 nAChR agonism as well as selectivity over 5-HT3A receptor antagonism. The general synthetic route to racemic spiroguanidines 1 is illustrated in Scheme 1. Key diamine intermediates 2 were synthesized in three steps from commercially available 3-quinuclidone, as has been described in the literature.8,12 For incorporation of the heterocyclic fragment, heteroaryl amines were converted to the isothiocyanates, or equivalents, as shown in Scheme 2. Coupling of isothiocyanates 3 with diamines 2, followed by N,N0 -diisopropylcarbodiimide-promoted cyclization afforded the desired spiroguanidines 1 in a convergent fashion (Scheme 3).13 Alternatively, dithioates 4 (synthesis shown in Scheme 4) could be coupled with diamine intermediate 2 to make the final spiroguanidine products 1 in a single step (Scheme 3). Chiral chromatography provided pure enantiomers.14 SAR data for selected compounds is provided in Table 1. We screened for both a7 nAChR agonism and 5-HT3A antagonism in Ca2+-based FLIPR (Fluorescence Image Plate Reader) assays.15,16 The relationship between these two values is reported for each compound as the FLIPR selectivity ratio. Earlier results demonstrated that a nitrogen atom was required at the 2-position of the heterocycle, ortho to the amino substituent from the guanidine (e.g., carbamimidate BMS-902483 in Fig. 1).8a With this in mind, diamine 2 (R = H) was used in the synthesis of various azaheterocyclic spiroguanidines having the ortho-aza orientation (examples 6–21). Monocyclic azaheterocycles were initially incorporated into analogs, including pyridine 6 and diazines 7– 10, which provided only modest FLIPR potency (P140 nM). Data on this cohort (examples 6–10) did suggest that substitution meta to the guanidine amine afforded a desirable loss in 5-HT3A affinity (examples 6–8 versus 9–10).

NH2

Het

Scheme 4. Representative synthesis of dithioates. (a) sodium hydroxide (10 N); carbon disulfide; sodium hydroxide (10 N); iodomethane, DMF, 0–23 °C.

N

Scheme 1. Synthetic route to spiroguanidines 1.

Het

N

Scheme 3. Two parallel synthetic routes to spiroguanidines 1. (a) Cs2CO3, DMF or MeCN, 23 °C; (b) N,N0 -diisopropylcarbodiimide, DMF or MeCN, 23–85 °C; (c) Cs2CO3, DMF, 23–85 °C.

Fig. 1. Examples of known a7 nAChR ligands.

C

NH2 2

BMS-902483

AZD-0328

MeS

NH

N

HN

C

3

2 R

N

S

NH2

AR-R17779

N

R NH

or

Het

N N

Scheme 2. Representative synthesis of isothiocyanate intermediates. (a) 1,10 thiocarbonyldiimidazole or 1,10 -thiocarbonyldipyridin-2(1H)-one.

6,6-Fused diazine-derived analogs 11–12 were similarly potent in the a7 nAChR assay regardless of ring geometry; however, quinazoline 12 provided a 5-HT3A receptor selectivity advantage. Preservation of this preferred ring geometry, coupled with ring contraction, provided a significant boost in both potency and selectivity as illustrated by 6,5-fused pyrrolo[1,2-f][1,2,4]triazine 13.8b Changes in both a7 nAChR potency and 5-HT3A receptor affinity were noted when the spiroguanidine connection was transferred from the 6-membered aryl ring and onto the five-membered heteroarene, exemplified by benzoxazole 14 and benzothiazole 16. Interestingly, simple substitutions on the benzene portion of the azaheterocycle led to significant changes in both potency and selectivity (examples 15, 17–21). For example, substitution at the 6-position afforded a significant increase in a7 nAChR selectivity via attenuation of the 5-HT3A receptor affinity (examples 15, 17– 19). Especially compelling was the comparison of examples 14 and 15, where a simple methyl replacement for hydrogen afforded over 250x increase in a7 nAChR selectivity versus the 5HT3A receptor. Conversely, 5- and 6-substituted analogs 20 and 21 were more potent at the 5-HT3A receptor (IC50 = 51 nM and 38 nM, respectively). These SAR trends strongly supported the hypothesis that an unfavorable steric interaction at the 5-HT3A binding site could be engaged to reduce affinity at that receptor. We explored methylation of the spiroguanidine amine in an effort to probe the impact of this vector on a7 nAChR and 5-HT3A receptor activities (Table 2). This change reduced a7 nAChR potency (example 16 versus 22), but the effect on 5-HT3A antagonism was more modest (examples 16 versus 22 and 19 versus 23). Homologation to N-ethyl blocked a7 nACh receptor binding and led to complete loss of affinity at that receptor (example 24). A structurally diverse set of spiroguanidine compounds was progressed into a concentration response electrophysiology (EP)12 patch clamp assay and evaluated for a7 nAChR agonist activity using HEK-293 cells that express rat a7 nAChR. Both potency and efficacy (Ymax – relative to both peak intensity and area under the curve for acetylcholine)15 data are shown for selected compounds in Table 3. Benzothiazole 16 (BMS-910731) was the most potent compound in EP studies. The binding potency of spiroguanidine 16 was subsequently measured as 240 nM by displacement of the radiolabeled a7 nAChR ligand, [125I]Tyr54-a-bungarotoxin.17 NMethylation led to a marked decrease in potency but little, if any, effect on efficacy (Table 3, examples 16 versus 22). Importantly, EP area Ymax values reflected partial agonism (<100%) and were consistent with FLIPR Ymax values that were measured between 50 and 80% for most analogs.

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M.D. Hill et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx Table 1

a7 nAChR and 5-HT3a FLIPR data for various azaheterocyclic spiroguanidines. HN N

Cmpda

H N

a7 EC50

5-HT3A IC50 (nM)b

(nM)b

Selectivity Ratiod

N

6

N

1200 (6)

860 (4)

0.72

5-HT3A IC50 (nM)b

Selectivity Ratiod

14

53 (2)

26 (4)

0.49

15

35 (5)

7400 (4)

210

26 (7)

26 (6)

1.0

17

150 (2)

18,000 (2)

120

18

880 (8)

32,000 (5)

36

19

56 (2)

32,000 (2)

570

20

750 (2)

51 (2)

0.068

21

120 (2)

38 (6)

0.32

N

7

N

290 (2)

620 (2)

2.1

O N

Cl

8

N N

(nM)b

N

O

Cl

N

HN N

a7 EC50

Cmpda

H N

990 (4)

5600 (3)

5.7

Me

16

S

c

N

Cl

9

N

1300 (4)

>100,000 (6)

>77

S N

N

Cl

OMe

10

N

140 (2)

6400 (2)

460

S N

N

OMe

N N

11

N

490 (2)

1400 (2)

2.9

S N

N

N

12

N

530 (2)

14,500 (5)

27

Cl

S N

N

OMe

13

N N

c

62 (4)

38,000 (2)

610

S N

N

Cl

Cl a b c d

Racemic product. Number of determinations in parentheses. Single enantiomer. Ratio = 5-HT3A IC50/a7 EC50.

Table 2

a7 nAChR and 5-HT3A receptor FLIPR data for alkylated-amine spiroguanidines. R N

N

H N

N S N

a b c d

5-HT3A IC50 (nM)b

Selectivity Ratiod

22 R = Me

170c (2)

43 (2)

0.25

23 R = Me

550 (2)

13,000 (2)

24

24 R = Et

>10,000 (5)

>12,000 (4)

>1

Cl S

N

a7 EC50 (nM)b

N

S

N

Cmpda

OMe

Racemic product. Number of determinations in parentheses. Single enantiomer. Ratio = 5-HT3A IC50/a7 EC50.

Reduced metabolic activation of the frontal cortices is a frequent finding in schizophrenia.18 Interestingly, a7 nAChR agonists are known to increase induction of immediate early genes c-fos and Arc, measures of cellular activation and plasticity, in the medial prefrontal cortex of rodents19 as well as mediate improved

performance in cognitive tasks that particularly engage this region.20 We examined the ability of 16 (0.3–10 mg/kg, sc) to induce c-fos and Arc in the rat medial prefrontal cortex as indicators for brain activity relevant to the a7 nicotinic mechanism (Table 4). In this study, the positive control, SSR180711,21 significantly induced

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M.D. Hill et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

Table 3

Supplementary data

a7 Concentration response EP data for select spiroguanidines.

a b

Compounda

a7 EP EC50 (nM, area)

Ymax (area, peak)

7 15 16b 17 22b

1100 500 200 1200 1200

44%, 58%, 78%, 33%, 67%,

11% 16% 29% 5% 25%

Racemic product. Single enantiomer.

Table 4 Normalized immediate early gene expression of 16.15

a b

Treatment

c-fosa

Vehicle (2 mL/kg, sc) 16, 0.3 mg/kg 16, 1 mg/kg 16, 3 mg/kg 16, 10 mg/kg SSR180711 10 mg/kg

1.0 ± 0.1 1.5 ± 0.1 1.1 ± 0.1 1.7 ± 0.3 1.9 ± 0.2 2.4 ± 0.4

Adjusted p valueb 0.48 1.0 0.087 0.018 0.00020

Arca 1.0 ± 0.1 1.2 ± 0.1 1.0 ± 0.1 1.5 ± 0.3 2.7 ± 0.6 3.9 ± 0.6

Adjusted p valueb 1.0 1.0 0.79 0.013 0.00010

Mean ± SEM (N = 8). Dunnett’s Multiple Comparison Test.

expression of c-fos and Arc (p = 0.00020 and 0.00010, respectively) compared with the vehicle group. Partial agonist 16 induced c-fos at the 3 mg/kg (p = 0.087) and 10 mg/kg (p = 0.018) doses with plasma concentrations = 130 nM (B/P = 4.0) and 460 nM (B/ P = 4.1), respectively. Arc induction occurred only at the highest dose, 10 mg/kg (p = 0.013).15 Expression of the endogenous control (b2-microglobulin; negative control) was not affected by any treatment. In summary, we identified a new series of spiroguanidinecontaining quinuclidines that provided a7 nAChR partial agonists with both monocyclic and fused bicyclic azaheteroaromatic groups appended to a spirocyclic amine. Fused 6,5-bicyclic heteroarenes provided a clear a7 nAChR potency advantage over monocyclic and fused 6,6-bicyclic systems. This SAR trend diverged from the pattern observed in the carbamimidate series.8 6,5-Bicyclic heteroarenes also functioned as a scaffold for elaboration that provided significant steric interactions with the 5-HT3A receptor and selectivity for the a7 nACh receptor. Furthermore, BMS-910731 induced immediate early genes c-fos and Arc in rat, in vivo findings relevant to the a7 nicotinic mechanism. Acknowledgments We acknowledge the Bioanalytical and Discovery Analytical Sciences group for chiral resolution of racemic mixtures to provide analogs 13, 16, and 22. We also acknowledge the Department of Discovery Synthesis for preparation of a chemical precursor.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12. 014. References and notes 1. (a) For reviews, please see: Saha S, McGrath J. Arch Gen Psychiatry. 2007;64:1123; (b) Horacek J, Bubenikova-Valesova V, Kopecek M, et al. CNS Drugs. 2006;20:389. 2. (a) For preclinical reports, please see: Sydserff S, Sutton EJ, Song D, et al. Biochem Pharmacol. 2009;78:880; (b) Castner SA, Smagin GN, Piser TM, et al. Biol Psychiatry. 2011;69:12; (c) Tatsumi R, Fujio M, Takanashi S, et al. J Med Chem. 2006;49:4374(d) For clinical efficacy data, please see: EnVivo Pharmaceuticals Presents Positive Comprehensive Phase 2b Study Results in Schizophrenia at American College of Neuropsychopharmacology Annual Meeting, http://www.envivopharma.com, EnVivo Pharmaceuticals Press Release, December 5; 2011.(e) Targacept Presents Statistically Significant Results for TC-5619 on Measures of Cognitive Dysfunction in Schizophrenia and Negative Symptoms of Schizophrenia, http:// www.targacept.com, Targacept, Inc. Press Release, April 7; 2011. 3. (a) For reviews, please see: Taly A, Corringer P-J, Guedin D, Lestage P, Changeux J-P. Nat Rev Drug Discov. 2009;8:733; (b) Leiser SC, Bowlby MR, Comery TA, Dunlop J. Pharmacol Ther. 2009;122:302; (c) Lightfoot AP, Kew JNC, Skidmore J. Prog Med Chem. 2008;46:131. 4. (a) Orr-Urtreger A, Göldner FM, Saeki M, et al. J Neurosci. 1997;17:9165; (b) Broide RS, Leslie FM. Mol Neurobiol. 1999;20:1. 5. Levin E, McClernon F, Rezvani A. Psychopharmacology. 2006;184:523. 6. (a) Wallace T, Porter R. Biochem Pharmacol. 2011;82:891; (b) Toyohara J, Hashimoto K. Open Med Chem Journal. 2010;4:37. 7. (a) Giniatullin R, Nistri A, Yakel JL. Trends Neurosci. 2005;28:381; (b) Broad LM, Sher E, Astles PC, Zwart R, O’Neill MJ. Drugs Future. 2007;32:161. 8. (a) Cook J, Zusi FC, McDonald IM, et al. J Med Chem. 2016. http://dx.doi.org/ 10.1021/acs.jmedchem.6b01506; (b) Hill MD, Fang H, King HD, et al. ACS Med. Chem. Lett. 2016. http://dx.doi.org/ 10.1021/acsmedchemlett.6b00471. 9. Guth BD, Rast G. Br J Pharmacol. 2010;159:22. 10. (a) For a description of off-target gastric side effects, please see: Gurley D, Lanthorn T. Neurosci Lett. 1998;247:107; (b) Briggs C, McKenna D. Neuropharmacology. 1998;37:1095; (c) Macor JE, Gurley D, Lanthorn T, et al. Bioorg Med Chem Lett. 2001;11:319; (d) Gallo-Torres H, Brinker A, Avigan M. Am J Gastroenterol. 2006;101:1080. 11. (a) Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC. Proc Natl Acad Sci USA. 2000;97:12329; (b) Fernandez D, Ghanta A, Kauffman GW, Sanguinetti MC. J Biol Chem. 2004;279:10120. 12. Mullen G, Napier J, Balestra M, et al. J Med Chem. 2000;43:4045. 13. The two-step conversion of isothiocyanates 3 and diamine 2 to spiroguanidines 1 can be achieved in one pot, without isolation acyclic urea 5, or two. 14. Chiral Supercritical Fluid Chromatography was used to separate enantiomers. 15. See Supplementary material for experimental details. 16. (a) Rudolf R, Mongillo M, Rizzuto R, Pozzan T. Nat Rev Mol Cell Biol. 2003;4:579; (b) González JE, Maher MP. Receptors Channel. 2002;8:283. 17. Moise L, Zeng H, Caffery P, Rogowski RS, Hawrot E. J Toxicol-Toxin Rev. 2002;21:293. 18. Parellada E, Catafau AM, Bernardo M, Lomeña F, Catarineu S, González-Monclús E. Biol Psychiatry. 1998;8:787. 19. Thomsen MS, Hay-Schmidt A, Hansen HH, Mikkelsen JD. Cereb Cortex. 2010;20:2092. 20. (a) McLean SL, Idris NF, Grayson B, et al. J Psychopharmacol. 2012;26:1265; (b) Birrell JM, Brown VJ. J Neurosci. 2000;20:4320. 21. Kristensen SE, Thomsen MS, Hansen HH, Timmermann DB, Hay-Schmidt A, Mikkelsen JD. Neurosci Lett. 2007;418:154.

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