Use of Structure-Based Design to Discover a Potent, Selective, In

Van de Water , A.; D'Aubioul , J.; Van Gerven , W.; De Chaffoy de Courcelles , D.; Freyne , E.; Xhonneux , R.; Reneman , R. S.; Janssen , P. A. J. Car...
0 downloads 0 Views 3MB Size
Download PDF
ARTICLE pubs.acs.org/jmc

Use of Structure-Based Design to Discover a Potent, Selective, In Vivo Active Phosphodiesterase 10A Inhibitor Lead Series for the Treatment of Schizophrenia Christopher J. Helal,* Zhijun Kang, Xinjun Hou, Jayvardhan Pandit, Thomas A. Chappie, John M. Humphrey, Eric S. Marr, Kimberly F. Fennell, Lois K. Chenard, Carol Fox, Christopher J. Schmidt, Robert D. Williams, Douglas S. Chapin, Judith Siuciak, Lorraine Lebel, Frank Menniti, Julia Cianfrogna, Kari R. Fonseca, Frederick R. Nelson, Rebecca O’Connor, Mary MacDougall, Laura McDowell, and Spiros Liras Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States

bS Supporting Information ABSTRACT: Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.

’ INTRODUCTION Phosphodiesterases (PDEs) constitute a family of enzymes that hydrolyze the ubiquitous intracellular messenger molecules cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), a process that regulates the activity of these molecules which have a role in essentially all physiological functions.1 The PDEs are divided into 11 families, with members of a given family demonstrating similar substrate specificity as well as inhibition by pharmacological tools. The central role played by PDEs in regulating physiological function has resulted in extensive efforts to identify inhibitors specific for a PDE family. This effort has elucidated the role of these enzymes in various biological processes to identify therapeutic opportunities among this class of enzymes. Central to the generation of specific inhibitors is the ability to determine the three-dimensional structure of PDEs via X-ray crystallography that can enable a guided approach to inhibitor design and optimization.2,3 Phosphodiesterase 10A (PDE10A), a phosophodiesterase that hydrolyzes both cGMP and cAMP, is expressed highly in the medium spiny neurons of the striatum.4 Recent work has shown that blockade of PDE10A with selective inhibitors increases striatal cGMP and phosphorylated CREB, a downstream marker of cAMP production.3,5 In conditioned avoidance responding (CAR), an animal model predictive of drug antipsychotic activity, PDE10A inhibitors produce a dose-dependent inhibition, an effect which is absent in PDE10A knockout mice, thus supporting this activity being specifically due to PDE10A inhibition.5 With an interest in being able to examine the effect of a PDE10A inhibitor in a clinical setting, we sought to bring forward r 2011 American Chemical Society

a number of structurally diverse chemical series that would increase the chances of finding a compound with the appropriate in vivo potency, pharmacokinetic properties, and safety. In particular, we sought a compound with PDE10A IC50 < 10 nM and low to moderate intrinsic human liver microsomal clearance (HLM Cl,int) (90% identical in the catalytic domain, and the numbering of the human residues is simply (10) from the corresponding rat residues, for example, Gln716(human) corresponds to Gln726(rat).

6,7-dimethoxy groups form a bidentate interaction with the NH of Gln-716, a conserved amino acid residue in all PDEs that is central to the binding of endogenous cyclic nucleotides; 2) the quinazoline ring sits within a hydrophobic clamp formed by Phe-719 on the top and Phe-686 on the bottom; 3) the phenyl group of the tetrahydroisoquinoline projects out of the hydrophobic pocket and interacts with the hydrophobic wall formed by Phe-719, Val-723, and Phe-629; 4) N-1 of the quinazoline interacts with the OH of Tyr-514 via a bridging water molecule; N-3 accepts a hydrogen bond from a water that interacts with the hydration sphere of the catalytic zinc and magnesium cations.8 It should be noted that the piperazine sits outside of the catalytic site, presumably exposed to solvent. In comparison, triarylimidazole 2 had PDE10A IC50 = 35 nM and showed weak activity against PDE3 (PDE3A = 11% inhibition @ 1 μM; PDE3B = 18% inhibition @ 10 μM).3b,5 Examination of

its cocrystal structure with the PDE10A catalytic domain revealed a possible basis for this selectivity, as it showed a binding interaction that is markedly different, not just from PDE10A inhibitors, but from other PDE inhibitors in general (Figure 3). Most importantly, the thiophene occupied a lipophilic pocket near the entrance of the hydrophobic cleft that defines the substrate binding site all PDEs. A comparison of the known crystal structures and a profiling of all PDEs binding sites sequence similarity show that this pocket is unique to PDE10A among all 21 PDEs in the family and thus may explain the high selectivity demonstrated by this series.3b Additionally, the imidazole accepts a hydrogen bond at the top of the pocket from the OH of Tyr-683 which accepts a hydrogen bond from the carboxamide amine of the conserved Gln-716 side chain. The other amide NH of the Gln-716 side chain forms a hydrogen bond with a buried water molecule. One 4-methoxyphenyl interacts with the Gln-716 amide side chain through van der Waals stacking contact. The second 4-methoxyphenyl makes a face-edge π interaction with Phe-719. Considering the difference in PDE3A/B selectivity profiles of quinazoline 1 and triaryl imidazole 2, we hypothesized that chemotypes like 1 could be made highly selective by accessing the same “selectivity pocket” that the thienylimidazole portion of 2 occupied. In particular, we sought to form an H-bond with Tyr693 and to fill the adjacent lipophilic pocket. Based upon the overlapping X-ray crystal structures of 1 and 2, variation of the 6-methoxy group of 1 appeared to allow access to the selectivity pocket. Also, considering the necessary orthogonal arrangement between the quinazoline and the desired group in the selectivity pocket, we reasoned that alkyl linkers would provide the optimal flexibility and thus focused efforts on these. An additional concern in the design process was the overall physical properties of subsequent compounds that would increase in molecular weight (MW) and potentially topological polar surface area (TPSA), thus resulting in limited brain penetration. Considering that 1 had MW = 483 and TPSA = 97 Å2, suggested that 6-methoxy derivatives could easily have MW >500 and TPSA >100 Å2.9 Thus, an alternative amine to the sulfonamide tetrahydroisoquinoline (STHIQ) in 1 was sought 4537

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547

Journal of Medicinal Chemistry Scheme 1. Comparison of Properties of 1 and 3

ARTICLE

Table 1. 6-Alkoxy SAR

Figure 4. Processes to generate a virtual library targeted to the PDE10A selectivity pocket.

with similar potency but reduced MW and TPSA. As mentioned previously,3a a chemical library approach to vary the amine in 1 was carried out and one of the amines that was found to afford reasonable potency with significantly lower MW and TPSA than the STHIQ was the 3-phenylpiperidine in compound 3 (Scheme 1).10 Based upon the modeled binding of 3 in the PDE10A X-ray structure, similar interactions were made as 1 with an improved ability of the 3-phenylpiperidine to form an edge-face π interaction with Phe-719. It was this amine that was thus used as part of the template for initial optimization work. To evaluate the optimal linker length and the preferred group to fill the selectivity pocket, we built a virtual library of 2500 compounds derived from the in silico coupling of all available alcohols and alkyl halides available in our in-house compound file with the quinazoline 6-hydroxy group, with a particular interest in those compounds containing hydrogen-bond acceptors (HBA) that could interact with Tyr-683 (Figure 4). We docked this virtual library into the PDE10A cocrystal structure of 1, with the quinazoline core constrained with a harmonic force to a position as in the X-ray structure of 1.11 Each molecule was docked 50 times with the piece-wise linear potential11 and rescored with an in-house scoring function.12 The best scored conformation was kept for each compound. The high scoring compounds were prioritized by the rescoring energy and visually examined, regardless of the physicochemical properties of the

compounds, to allow for identification of desirable binding motifs. This exercise suggested that 2-carbon linkers attached to various H-bond accepting groups would allow for optimal access to the selectivity pocket along with H-bond interactions with Tyr-683. All enumerated compounds were next filtered by calculated properties favoring brain penetration (topological polar surface area 5-fold as compared to being 19

MDR BA/AB

0.7

>10

Figure 8. Benzimidazole analog from parallel chemistry efforts.

linker, was potent (PDE10A IC50 = 10 nM) and showed high selectivity versus PDE3A (Figure 8). Compounds such as 16 and 19 are highly potent and selective PDE10A inhibitors, but their ligand efficiency (LE), a measure of potency per atom,15 is reduced as compared to 3. The increases in molecular weight and lipophilicity (cLogD) result in higher HLM Cl,int and significant in vitro Pgp efflux (Table 2). We hypothesized that the quinoline interaction in the selectivity pocket was the major potency and selectivity “anchor” for 16, possibly rendering the phenylpiperidine superfluous, and thus targeted truncated analogs with more desirable physicochemical properties to test this hypothesis. We first prepared 20, which showed a loss in potency but a significant improvement in LE (Figure 9). This result supported the hypothesis that the phenylpiperidine was not providing optimal binding efficiency in 16, and compound 21 became the next target. Hydrogenolysis of commercially available 2,4-dichloro-6,7dimethoxyquinazoline gave 22 (Scheme 4). Selective acidic demethylation resulted in 7-hydroxy-6-methoxyquinazoline 23, as opposed to the desired 6-hydroxy-7-methoxyquinazoline. This unexpected switch in regiochemistry did not allow for access to the targeted 21. This alternative regioisomer was carried forward

Figure 9. Improvements in LE suggest new SAR directions.

into a Mitsunobu coupling with 2-quinolylethanol 18 and afforded 24, which has the quinazoline nitrogens in different positions than previous quinazoline analogs. Compound 24 showed excellent potency, selectivity, ligand efficiency, low HLM Cl, int, no Pgp efflux, and moderate permeability. The crystal structure of 24 bound to PDE10A showed that the quinoline group interaction in the selectivity pocket was completely identical to that seen in the structure of 16 (Figure 10), confirming the role of this group as a binding ’anchor’. It should be pointed out that the water molecules that interact with the quinazoline nitrogens in 1 were not observed in the X-ray crystal structure of 24. This observation suggests that these bridging water interactions may be less critical when the selectivity pocket is efficiently occupied. No activity was observed when 24 was tested for its ability to increase striatal cGMP in mouse at 5.6 mg/kg, sc. It was presumed that the moderate in vitro permeability could be translating into less than optimal in vivo brain penetration. Additionally, the necessary exposure may have been limited by high rodent specific clearance (rat microsomal Cl,int = 183 mL/min/kg). 4540

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547

Journal of Medicinal Chemistry Scheme 4a

ARTICLE

Scheme 5a

a

Reaction conditions: (a) Pd/C, TEA, MeOH, H2 (40 psi), 65%; (b) MeSO3H, 120-145 °C, 57%; (c) di-tert-butylazodicarboxylate, triphenylphosphine, 18, THF, 38%. L-methionine,

Reaction conditions: (a) NaOH, H2O, 100 °C, 99%; (b) H2SO4, MeOH, reflux, 96%; (c) BnBr, Cs 2 CO 3 , DMSO, 94%; (d) Fe, ammonium chloride, MeOH, H2O, 90 °C, 86%; (e) LiOH, MeOH, H2O, 75 °C, 93%; (f) formamidine acetate, MeOCH2 CH 2OH, 130 °C, 94%; (g) POCl3 , 120 °C, 3 h, 99%; (h) HNMe2, DIPEA, IPA, 99%; (i) TFA, anisole, 75 °C, 6595%; (j) di-tert-butylazodicarboxylate, triphenylphosphine, 18, THF, 20-60%; (k) 3-(Et2B)pyridine, Pd2(dba)3-CHCl3, Cs2CO3, dioxane, 100 °C, 4070%. a

Scheme 6a

Figure 10. Overlap of 24 (cyan, PDB ID 3QPN) and 16 (magenta).

We sought to address the challenge of balancing permeability and clearance by variations on the quinazoline core. In particular, based upon the X-ray crystal structure, and the knowledge that 3-phenylpiperidine was tolerated as in 16, structural modifications at the quinazoline 4-position were pursued (Schemes 5 and 6). Three series of analogs were made. A comparison of representative compounds from the amino (25) and aryl (26) class, as well as quinazolinone (27), is shown in Table 3. The dimethylamino quinazoline 25 and the quinazolinone 27 had similar profiles with PDE10A IC50 = 14 nM and 12 nM, respectively, high permeability with no Pgp efflux, and high HLM, Cl, int. The 3-pyridyl quinazoline 26 afforded PDE10A IC50 = 5 nM, low human microsomal clearance, no Pgp efflux, with low permeability. All compounds showed the ability to increase brain cGMP in mouse in vivo to differing degrees (See Supporting Information). On the basis of the low human

Reaction conditions: (a) (MeO)3CH, 105 °C; MeNH2, toluene, 80 °C, 86% yield; (b) TFA, anisole, 75 °C, 95% yield; (c) di-tert-butylazodicarboxylate, triphenylphosphine, 18, THF, r.t., 67% yield. a

microsomal clearance of 26, which would allow for potentially improved in vivo half-life, it was tested in the mouse CAR model, where clear efficacy was observed with an ED50 = 3.2 mg/kg, sc, in wild type mice (Figure 11). No effect was seen in PDE10A knockout mice, which demonstrated that the efficacy in wild type mice was specific to PDE10A inhibition.4b 4541

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547

Journal of Medicinal Chemistry

ARTICLE

Table 3. 4-Position Variations 25

27

PDE10A IC50 (nM)

14

5

12

HLM, CI, int (mL/min/kg)

178

12 using 1 N sodium hydroxide. The biphasic mixture was extracted with chloroform, the organic layer was washed with brine, was dried over MgSO4, was filtered, and was concentrated. Purification by silica gel chromatography (chloroformmethanol, 75:1) gave 440 mg (86% yield) of the title compound. 1H NMR (400 MHz, CDCl3) δ (ppm) = 8.52 (s, 1H), 7.44 (m, 2H), 7.36 (m, 2H), 7.28 (m, 1H), 7.21 (s, 1H), 7.17 (s, 1H), 5.26 (s, 2H), 4.01 (s, 3H), 3.06 (s, 6H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 163.26, 154.77, 153.14, 149.25, 146.30, 136.60, 128.97, 128.28, 127.18, 110.39, 107.94, 107.73, 71.46, 56.34, 41.66. MS (AP/CI): 310.1 (M þ H)þ. Step I: 4-(Dimethylamino)-7-methoxyquinazolin-6-ol. 6-(Benzyloxy)7-methoxy-N,N-dimethylquinazolin-4-amine (410 mg, 1.33 mmol) and anisole (2.9 mL, 26.6 mmol) in trifluoroacetic acid (25 mL) were heated at 75 °C for 24 h. The solvent was removed in vacuo and the residue was purified by silica gel chromatography (chloroformmethanol, 10:1) to afford the title compound in 99% yield (290 mg). 1H NMR (400 MHz, CD3OD/CDCl3) δ (ppm) = 8.40 (s, 1H), 7.59 (s, 1H), 7.23 (s, 1H), 4.02 (s, 3H), 3.56 (s, 6H); 13C NMR (100 MHz, CD3OD/CDCl3) δ (ppm) = 161.27, 155.87, 147.37, 145.60, 136.02, 110.07, 107.36, 99.55, 56.51, 42.36. MS (AP/CI): 220.2 (M þ H)þ. 7-Methoxy-N,N-dimethyl-6-(2-quinolin-2-ylethoxy)quinazolin-4amine (25). Following the procedure for compound 16 using 4(dimethylamino)-7-methoxyquinazolin-6-ol as starting material, the desired product was obtained in 32% yield (86 mg) following silica gel chromatography. 1H NMR (400 MHz, CDCl3) δ (ppm) = 8.52 (s, 1H), 8.07 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.67 (m, 1H), 7.48 (m, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.16 (s, 1H), 4.59 (t, i = 7.1 Hz, 2H), 3.94 (s, 3H), 3.53 (t, J = 7.1 Hz,

ARTICLE

2H), 3.20 (s, 6H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 163.35, 158.99, 154.66, 153.09, 149.23, 148.20, 146.93, 136.63, 129.76, 129.02, 127.83, 127.20, 126.31, 122.4, 110.61, 107.69, 106.78, 68.89, 6.26, 41.90, 38.77. MS (AP/CI): 375.3 (M þ H)þ. Step K: 6-(Benzyloxy)-7-methoxy-4-(pyridin-3-yl)quinazoline. 6-(Benzyloxy)-4-chloro-7-methoxyquinazoline (1 g, 3.3 mmol) was mixed with 3-(1,3,2-dioxaborinan-2-yl)pyridine (0.65 g, 4 mmol), Pd2(dba)3-CHCl3 (72 mg, 0.07 mmol), tricyclohexyl phosphine (56 mg, 0.2 mmol), and cesium carbonate (1.6 g, 5 mmol) in dioxane (10 mL). The mixture was heated to 100 °C for 24 h, was cooled to room temperature, was concentrated in vacuo, was diluted with 1 N sodium hydroxide and water (1:1), and was extracted with chloroform, and the organic layer was washed with brine and was dried over MgSO4, was filtered, and was concentrated in vacuo. Purification by silica gel chromatography (ethyl acetatehexanes-methanol 8:1:0 to 8:1:0.5) afforded 1 g (88% yield) of the title compound. 1H NMR (400 MHz, CDCl3) δ (ppm) = 9.17 (s, 1H), 8.90 (d, J = 1.7 Hz, 1H), 8.78 (dd, J = 1.7, 5.0 Hz, 1H), 7.82 (dt, J = 2.1, 7.9 Hz, 1H), 7.437.31 (m, 6H), 7.20 (s, 1H), 5.19 (s, 2H), 4.08 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 162.09, 156.73, 153.85, 150.82, 150.40, 149.76, 149.51, 137.19, 135.94, 133.67, 129.04, 128.55, 127.48, 123.71, 118.81, 107.50, 105.74, 71.18, 56.72. MS (AP/CI): 344.1 (M þ H)þ. Step I: 7-Methoxy-4-(pyridin-3-yl)quinazolin-6-ol. 6-(Benzyloxy)-7methoxy-4-(pyridin-3-yl)quinazoline (1 g, 2.9 mmol) and anisole (6.3 g, 58 mmol) in trifluoroacetic acid (40 mL) was heated at 75 °C for 24 h. After the mixture was cooled to room temperature and concentration in vacuo, the residue was purified by silica gel chromatography (chloroform methanol, 30:1) to afford 0.73 g (99% yield) of the title compound. 1H NMR (400 MHz, CD3OD/CDCl3) δ (ppm) = 9.07 (s, 1H), 8.96 (d, J = 1.7 Hz, 1H), 8.77 (dd, J = 1.7, 5.0 Hz, 1H), 8.31 (m, 1H), 7.73 (dd, J = 5.0, 7.9 Hz, 1H), 7.39 (s, 1H), 7.27 (s, 1H), 4.09 (s, 3H); 13C NMR (100 MHz, CD3OD/CDCl3) δ (ppm) = 161.07, 156.88, 151.64, 149.92, 149.05, 148.44, 148.03, 139.51, 134.29, 124.71, 119.62, 106.54, 105.88, 56.34. MS (AP/CI): 254.1 (M þ H)þ. 7-Methoxy-4-pyridin-3-yl-6-(2-quinolin-2-ylethoxy)quinazoline (26). Following the procedure for compound 16 using 7-methoxy-4-(pyridin3-yl)quinazolin-6-ol as starting material, the title compound was obtained in 63% yield. 1H NMR (400 MHz, CDCl3) δ (ppm) = 9.17 (s, 1H), 8.99 (brs, 1H), 8.74 (brs, 1H), 8.06 (m, 2H), 7.96 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.66 (m, 1H), 7.47 (m, 2H), 7.40 (d, J = 8.7, 1H), 7.35 (s, 1H), 7.28 (s, 1H), 4.52 (t, J = 6.6 Hz, 2H), 4.01 (s, 3H), 3.52 (t, J = 6.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 38.5, 56.6, 68.6, 104.6, 107.4, 119.0, 122.3, 123.9, 126.4, 127.2, 127.8, 129.0, 129.8, 133.8, 136.7, 137.2, 148.1, 149.5, 150.3, 150.4, 150.9, 153.8, 156.6, 158.5, 162.1. MS (AP/CI): 409.0 (M þ H)þ.

Preparation of Key Intermediates in Scheme 6 Toward Preparation of Compound 27. Step A: 6-(Benzyloxy)-7-methoxy3-methylquinazolin-4(3H)-one. A solution of 2-amino-5-(benzyloxy)4-methoxybenzoic acid (Scheme 5, Step E) (100 mg, 0.37 mmol) in trimethyl orthoformate (1 mL, 9 mmol) was heated at 105 °C for 2 h. The solvent was removed in vacuo, dry toluene was added, followed by methylamine (2 M in THF, 1.83 mL). The mixture was heated to 80 °C for 24 h, the solvent was removed in vacuo, and the residue was purified by silica gel chromatography (ethyl acetatemethanol, 50:1) to afford 99 mg (91% yield) of the title compound. 1H NMR (400 MHz, CDCl3) δ (ppm) = 7.94 (s, 1H), 7.68 (s, 1H), 7.47 (m, 2H), 7.387.25 (m, 3H), 7.09 (s, 1H), 5.23 (s, 2H), 3.97 (s, 3H), 3.55 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 161.12, 155.50, 148.69, 145.82, 144.88, 136.36, 128.86, 128.36, 127.79, 115.50, 108.20, 107.45, 71.19, 56.47, 34.28. MS (ESþ): 297.5 (M þ H)þ. Step B: 6-Hydroxy-7-methoxy-3-methylquinazolin-4(3H)-one. 6-(Benzyloxy)-7-methoxy-3-methylquinazolin-4(3H)-one was converted to the title compound in 95% yield (58 mg) using the debenzylation conditions as detailed for Scheme 5, Step I. 1H NMR (400 MHz, 4545

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547

Journal of Medicinal Chemistry CD3OD/CDCl3) δ (ppm) = 8.03 (s, 1H), 7.50 (s, 1H), 7.03 (s, 1H), 3.96 (s, 1H), 3.54 (s, 1H); 13C NMR (100 MHz, CD3OD/CDCl3) δ (ppm) = 161.58, 154.57, 147.46, 145.70, 143.49, 115.54, 109.09, 107.10, 55.93, 33.87. MS (ESþ): 207.4 (M þ H)þ. 7-Methoxy-3-methyl-6-(2-quinolin-2-ylethoxy)quinazolin-4(3H)-one (27). 6-Hydroxy-7-methoxy-3-methylquinazolin-4(3H)-one was converted to the title compound in 67% yield (65 mg) using the coupling conditions as detailed for compound 16. 1H NMR (400 MHz, CDCl3) δ (ppm) = 8.07 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.65 (m, 1H), 7.47 (m, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.05 (s, 1H), 4.63 (t, J = 6.9 Hz, 2H), 3.92 (s, 3H), 3.563.53 (m, 5H); 13C NMR (100 MHz, CDCl3) δ (ppm) = 161.12, 158.87, 155.36, 148.89, 148.22, 145.75, 144.79, 136.52, 129.67, 129.23, 127.70, 127.18, 126.21, 122.20, 115.59, 108.18, 107.20, 68.59, 56.41, 38.64, 34.26; MS (ESþ): 362.5 (M þ H)þ.

Biology Experimental Data. PDE10 Enzyme Assay Protocol. PDE10A was generated from the full-length recombinant rat clone transfected into Sf9 cells. The enzyme was extracted from cell pellets in lysis buffer (20 mM Tris, 2 mM benzamidine, 1.0 mM Na2EDTA, 0.25 M sucrose, 100 μM PMSF, pH 7.5 at room temperature) and stored frozen at 80 °C. PDE activity was measured using a plate based Scintillation Proximity Assay (SPA) modified from an Amersham Biosciences protocol (TRKQ7090). The Km of the PDE10A preparation was experimentally determined to be 24 nM at room temperature. For competitive enzyme inhibition assays, the substrate [3H]cAMP concentration (New England Nuclear NET27500) was held at 20 nM for conditions to be at or below the Km of the enzyme. The concentration of enzyme was adjusted to convert less than 10% of available substrate to end product during the assay. Compounds were initially dissolved in DMSO and diluted such that the final DMSO assay concentration was 3%. Following the addition of the test agents and [3H] cAMP, enzyme was added in buffer containing 50 mM Tris and 1.3 mM MgCl2 (pH 7.5) to a final volume of 50 ul. The incubation was allowed to proceed for 30 min at room temperature before the addition of 20 ul of PDE SPA beads (Amersham Biosciences; RPNQ0150) at 0.2 mg/well to stop the reaction. Plates were allowed to stand 10 to 12 h before counting in a Trilux plate reader. IC50s were calculated after the subtraction of background as determined by addition of 10 μM papaverine.

’ ASSOCIATED CONTENT

bS

Supporting Information. PDE selectivity data for 16, 24, 26, and 27, X-ray crystal structure experimental data, PDE10A assay data with standard error, effects of compounds 25, 26, and 27 on striatal cGMP levels, and effect of compound 26 on conditioned avoidance responding (CAR) in wild type and PDE10A knockout mice.This material is available free of charge via the Internet at http://pubs.acs.org.

Accession Codes

Information has been deposited with Protein Databank as the following codes: 1, 2O8H; 2, 3HQW; 13, 3QPO; 16, 3QPP; 24, 3QPN.

’ AUTHOR INFORMATION Corresponding Author

*Tel: (860) 715-5064. Fax: (860) 686-5291. E-mail: chris.j. helal@pfizer.com. Address: MS 8220-4326, Eastern Point Road, Groton, CT 06340.

ARTICLE

’ ABBREVIATIONS USED ((), indicates a racemic compound; Gln, glutamine; ADME, Absorption, Distribution, Metabolism, Excretion; CAR, conditioned avoidance responding; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; HBA, hydrogen bond acceptor; HLM Cl,int, human liver microsomal intrinsic clearance; HPLC, high performance liquid chromatography; MDR BA/AB, ratio of Basil to Apical/Apical to Basal permeability in a canine kidney cell line overexpressing the human multidrug resistant transporter (aka P-glycoprotein) with flux measured as 106 cm/sec; MW, molecular weight; PDE, phosphodiesterase; Pgp, p-glycoprotein; Phe, phenylalanine; STHIQ, sulfonyl tetrahydroisoquinoline; TPSA, topological polar surface area; Tyr, tyrosine; Val, valine ’ REFERENCES (1) Jeon, Y. H.; Heo, Y. S.; Kim, C. M.; Hyun, Y. L.; Lee, T. G.; Ro, S.; Cho, J. M. Phosphodiesterase: Overview of Protein Structures, Potential Therapeutic Applications and Recent Progress in Drug Development. Cell. Mol. Life Sci. 2005, 62, 1198–1220. (2) (a) Manallack, D. T.; Hughes, R. A.; Thompson, P. E. The Next Generation of Phosphodiesterase Inhibitors: Structural Clues to Ligand and Substrate Selectivity of Phosphodiesterases. J. Med. Chem. 2005, 48, 3449–3462. (b) Card, G. L.; England, B. P.; Suzuki, Y.; Fong, D.; Powell, B.; Lee, B.; Luu, C.; Tabrizizad, M.; Gillette, S.; Ibrahim, P. N.; Artis, D. R.; Bollag, G.; Milburn, M. V.; Kim, S.-H.; Schlessinger, J.; Zhang, K. Y. J. Structural Basis for the Activity of Drugs That Inhibit Phosphodiesterases. Structure (Cambridge, MA, U. S. A.) 2004, 12, 2233–2247. (3) (a) Chappie, T. A.; Humphrey, J. M.; Allen, M. P.; Estep, K. G.; Fox, C. B.; Lebel, L. A.; Liras, S.; Marr, E. S.; Menniti, F. S.; Pandit, J.; Schmidt, C. J.; Tu, M.; Williams, R. D.; Yang, F. V. Discovery of a Series of 6,7-Dimethoxy-4-pyrrolidylquinazoline PDE10A Inhibitors. J. Med. Chem. 2007, 50, 182–185. (b) Verhoest, P. R.; Chapin, D. S.; Corman, M.; Fonseca, K.; Harms, J. F.; Hou, X.; Marr, E. S.; Menniti, F. S.; Nelson, F.; O’Connor, R.; Pandit, J.; Proulx-LaFrance, C.; Schmidt, A. W.; Schmidt, C. J.; Suiciak, J. A.; Liras, S. Discovery of a Novel Class of Phosphodiesterase 10A Inhibitors and Identification of Clinical Candidate 2-[4-(1-Methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline (PF-2545920)(I) for the Treatment of Schizophrenia. J. Med. Chem. 2009, 52, 5188–5196. (c) Hoefgen, N.; Stange, H.; Schindler, R.; Lankau, H.-J.; Grunwald, C.; Langen, B.; Egerland, U.; Tremmel, P.; Pangalos, M. N.; Marquis, K. L.; Hage, T.; Harrison, B. L.; Malamas, M. S.; Brandon, N. J.; Kronbach, T. Discovery of Imidazo[1,5a]pyrido[3,2-e]pyrazines as a New Class of Phosphodiesterase 10A Inhibitors. J. Med. Chem. 2010, 53, 4399–4411. (4) (a) Seeger, T. F.; Bartlett, B.; Coskran, T. M.; Culp, J. S.; James, L. C.; Krull, D. L.; Lanfear, J.; Ryan, A. M.; Schmidt, C. J.; Strick, C. A.; Varghese, A. H.; Williams, R. D.; Wylie, P. G.; Menniti, F. S. Immunohistochemical Localization of PDE10A in the Rat Brain. Brain Res. 2003, 985, 113–126. (b) Schmidt, C. J.; Chapin, D. S.; Cianfrogna, J.; Corman, M. L.; Hajos, M.; Harms, J. F.; Hoffman, W. E.; Lebel, L. A.; McCarthy, S. A.; Nelson, F. R.; Proulx-LaFrance, C.; Majchrzak, M. J.; Ramirez, A. D.; Schmidt, K.; Seymour, P. A.; Siuciak, J. A.; Tingley, F. D., III; Williams, R. D.; Verhoest, P. R.; Menniti, F. S. Preclinical Characterization of Selective Phosphodiesterase 10A Inhibitors: A New Therapeutic Approach to the Treatment of Schizophrenia. J. Pharmacol. Exp. Ther. 2008, 325, 681–690. (5) (a) Siuciak, J. A.; Chapin, D. S.; Harms, J. F.; Lebel, L. A.; McCarthy, S. A.; Chambers, L.; Shrikhande, A.; Wong, S.; Menniti, F. S.; Schmidt, C. J. Inhibition of the Striatum-Enriched Phosphodiesterase PDE10A: A Novel Approach to the Treatment of Psychosis. Neuropharmacology 2006, 51, 386–396. (b) Strick, C. A.; Schmidt, C. J.; Menniti, F. S. PDE10A: A Striatum-Enriched, Dual-Substrate Phosphodiesterase. Cyclic Nucleotide Phosphodiesterases Health Dis. 2007, 237–254. (6) Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski, J. S.; Troutman, M. D.; de Morais, S. M. In vitro P-glycoprotein Assays to 4546

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547

Journal of Medicinal Chemistry

ARTICLE

Predict the In Vivo Interactions of P-Glycoprotein with Drugs in the Central Nervous System. Drug Metab. Dispos. 2008, 36, 268–275. (7) (a) Sugiyama, A.; Satoh, Y.; Hashimoto, K. Electropharmacologic Effects of A New Phosphodiesterase III Inhibitor, Toborinone (OPC-18790), Assessed in An In Vivo Canine Model. J. Cardiovasc. Pharmacol. 2001, 38, 268–277. (b) Van de Water, A.; D’Aubioul, J.; Van Gerven, W.; De Chaffoy de Courcelles, D.; Freyne, E.; Xhonneux, R.; Reneman, R. S.; Janssen, P. A. J. Cardiac and Hemodynamic Effects of Intravenous R 80122, A New Phosphodiesterase III Inhibitor, In Anesthetized and Awake Dogs. Arch. Int. Pharmacodyn. Ther. 1992, 316, 60–74. (8) Pandit, J. Crystal Structure of 30 ,50 -Cyclicnucleotide Phosphodiesterase (PDE10A) and Uses Thereof. U.S. Pat. Appl. Publ. 0202550, 2005. (9) (a) Gleeson, M. P. Generation of a Set of Simple, Interpretable ADMET Rules of Thumb. J. Med. Chem. 2008, 51, 817–834. (b) Clark, D. E. Rapid Calculation of Polar Molecular Surface Area and Its Application to the Prediction of Transport Phenomena. 2. Prediction of Blood-Brain Barrier Penetration. J. Pharm. Sci. 1999, 88, 815–821. (c) Doan, K. M. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. Passive Permeability and P-Glycoprotein-Mediated Efflux Differentiate Central Nervous System (CNS) and Non-CNS Marketed Drugs. J. Pharmacol. Exp. Ther. 2002, 303, 1029–1037. (10) Separation of enantiomers in a structurally related analog (p-methoxy phenyl) resulted in one enantiomer demonstrating significantly more PDE10 and PDE3A/B activity (>20) than the other. As such, the use of the racemic 3-phenylpiperidine was deemed feasible for assessing selectivity. (11) Gehlhaar, D. K.; Verkhivker, G. M.; Rejto, P. A.; Sherman, C. J.; Fogel, D. B.; Fogel, L. J.; Freer, S. T. Molecular Recognition of the Inhibitor AG-1343 by HIV-1 Protease: Conformationally Flexible Docking by Evolutionary Programming. Chem. Biol. 1995, 2, 317–324. (12) Marrone, T. J.; Luty, B. A.; Rose, P. W. Discovering HighAffinity Ligands from the Computationally Predicted Structures and Affinities of Small Molecules Bound to A Target: A Virtual Screening Approach. Perspect. Drug Discovery Des. 2000, 20, 209–230. (13) If all reagents were mixed together at the same time, products from the addition of 5 to the azodicarboxylate were observed with little product. See: Hughes, D. L. The Mitsunobu Reaction. Org. React. (Hoboken, NJ, U. S. A.) 1992, 42. (14) Helal, C. J. Heteroaromatic Quinoline Compounds as Phosphodiesterase Inhibitors, Their Preparation, Pharmaceutical Compositions, and Use in Treating CNS and Other Disorders.WO 2008/020302 A2, 2008. (15) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand Efficiency: A Useful Metric for Lead Selection. Drug Discovery Today 2004, 9, 430–431. (16) Lombardino, J. G. Pharmaceutical Imidazoles. Ger. Offen. DE 2155558, 1972.

4547

dx.doi.org/10.1021/jm2001508 |J. Med. Chem. 2011, 54, 4536–4547