Design, Synthesis, and Pharmacological Evaluation of 5,6-Disubstituted Pyridin-2(1H)-one Derivatives as Phosphodiesterase 10A (PDE10A) Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.5b01240

We report the design and synthesis of novel 5,6-diarylated pyridin-2(1H)-one derivatives as pharmacophoric PDE10A inhibitors. This highly potent molecular scaffold was developed from an inactive diarylpyridine-2-amine derivative 3b by extensive and systematic analogue synthesis and SAR analysis. Further optimization of the scaffold resulted in identification of pyridin-2(1H)-one 18b as a lead compound with good potency (IC₅₀ = 1.6 nM) and selectivity (>6000-fold) over other related PDEs but with a poor pharmacokinetic profile. Careful metabolite profiling of 18b revealed that poor systemic exposure in rats (C_max = 44 ng/mL; AUC_0-t = 359 ng·h/mL) at 10 mg/kg was due to the formation of O-glucuronide conjugate by phase 2 metabolism. The structure of the glucuronide metabolite was confirmed by retention time and LC-MS/MS fragmentation matching with the synthetic glucuronide 26. The problem of low exposure of 18b was effectively addressed by its conversion to an acetate prodrug 25b, which upon oral dosing resulted in an improved pharmacokinetic profile (C_max = 359 ng.h/mL; AUC_0-t = 2436 ng.h/mL) and a desirable brain to plasma ratio of 1.2. The prodrug 25b showed good efficacy in selected rodent models of psychosis.

Full text of DOI:10.1021/acs.jmedchem.5b01240

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Article
Design, Synthesis and Pharmacological Evaluation
of 5,6-Disubstituted Pyridin-2(1H)-one Derivatives
as Phosphodiesterase 10A (PDE10A) Antagonists
Abraham Thomas, V. S. Prasadarao Lingam, Dnyaneshwar H. Dahale, Vijay E. Rathi,
Yogesh B. Shingote, Rajni R. Thakur, Ajit S. Mindhe, Srinivas Kummari, Neelima
Khairatkar-Joshi, Malini Bajpai, Daisy M. Shah, Ratika S. Sapalya, Srinivas Gullapalli,
Praveen K. Gupta, Girish S. Gudi, Satyawan B. Jadhav, and Rambabu Pattem
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01240 • Publication Date (Web): 30 Sep 2015
Downloaded from http://pubs.acs.org on October 4, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Journal of Medicinal Chemistry
Design, Synthesis and Pharmacological Evaluation
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of 5,6ꢀDisubstituted Pyridinꢀ2(1H)ꢀone Derivatives
as Phosphodiesterase 10A (PDE10A) Antagonists
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V. S. Prasadarao Lingam, Dnyaneshwar H. Dahale, Vijay E. Rathi, Yogesh B. Shingote,
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Rajni R. Thakur, Ajit S. Mindhe, Srinivas Kummari, Neelima Khairatkar-Joshi, Malini
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Bajpai, Daisy M. Shah, Ratika S. Sapalya, Srinivas Gullapalli, Praveen K. Gupta, Girish S.
#
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#
1†
Gudi, Satyawan B. Jadhav, Rambabu Pattem, and Abraham Thomas
†
‡
#
Medicinal Chemistry Division, Pharmacology Division, Drug Metabolism and
Pharmacokinetics, Glenmark Research Centre, Aꢀ607, TTC Industrial Area, MIDC Mahape,
Navi Mumbai ꢀ 400 709, India
ABSTRACT: We report the design and synthesis of novel 5,6ꢀdiarylated pyridinꢀ2(1H)ꢀone
derivatives as pharmacophoric PDE10A inhibitors. This highly potent molecular scaffold was
developed from an inactive diarylpyridineꢀ2ꢀamine derivative 3b by extensive and systematic
analogue synthesis and SAR analysis. Further optimization of the scaffold resulted in
identification of pyridinꢀ2(1H)ꢀone 18b as a lead compound with good potency (IC : 1.6 nM)
5
0
and selectivity (> 6000 fold) over other related PDEs but with a poor pharmacokinetic profile.
Careful metabolite profiling of 18b revealed that poor systemic exposure in rats (Cmax: 44
ng/mL; AUC(0ꢀt): 359 ng.h/mL) at 10 mg/kg was due to the formation of Oꢀglucuronide
conjugate by phase 2 metabolism. The structure of the glucuronide metabolite was confirmed by
retention time and LCꢀMS/MS fragmentation matching with the synthetic glucuronide 26. The
problem of low exposure of 18b was effectively addressed by its conversion to an acetate
1
To whom correspondence should be addressed. Phone: +91ꢀ226772ꢀ0000. Fax: +91ꢀ2222ꢀ
2778ꢀ2525. Eꢀmail: abraham.thomas@glenmarkpharma.com
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prodrug 25b, which upon oral dosing resulted in an improved pharmacokinetic profile (Cmax: 359
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ng.h/mL; AUC(0ꢀt): 2436 ng.h/mL) and a desirable brain to plasma ratio of 1.2. The prodrug 25b
showed good efficacy in selected rodent models of psychosis.
INTRODUCTION
Phosphodiesterase 10A (PDE10A) is a dual substrate specific enzyme that hydrolyzes both
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′,5′ꢀcyclic adenosine monophosphate (cAMP) and 3′,5′ꢀcyclic guanosine monophosphate
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cGMP) to the corresponding inactive phosphates. It has a higher affinity for cAMP (K = 0.05
m
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µM) than for cGMP (K = 3.0 µM). PDE10A enzyme has been considered as a potential
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drugable target for the treatment of several central nervous system (CNS) disorders. The
enzyme is highly expressed in certain areas of the brain linked to CNS disorders including
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schizophrenia and Huntington’s disease. Schizophrenia is characterized by disordered thinking
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and behaviour and the patients, in general, suffer from delusions and hallucinations.
Huntington’s disease is a neurodegenerative disease characterized by abnormalities mainly in
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cognition and movement. The currently available therapy using typical antipsychotics (D2
receptor antagonists, e.g. haloperidol, fluphenazine) and atypical antipsychotics (D2/5HT2A
antagonists, e.g. quetiapine, clozapine) are effective in treating only positive symptoms. These
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drugs are ineffective against negative symptoms and cognitive deficits of schizophrenia. These
drugs are also associated with unwanted side effects such as weight gain, diabetes and
cardiovascular diseases. Recent studies have shown that PDE10A could modulate both
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dopaminergic and glutamatergic signaling pathways of the brain. Thus, PDE10A inhibitors have
the potential to address a high unmet medical need in treating negative and cognitive symptoms
along with good efficacy on positive symptoms of the disease with fewer side effects.
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In the previous studies with this target, we and others had identified diverse structural motifs
that are efficient inhibitors of PDE10A. A large number of small molecule PDE10A inhibitors
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are now reported in the literature and are in various stages of preclinical and clinical studies.
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Pfizer’s wellꢀstudied molecule MPꢀ10 (PFꢀ02545920) and Omeros’ molecule OMS824 are
currently in phase 2 clinical trials for treating positive and negative symptoms associated with
schizophrenia and Huntington's disease. Omeros’ proprietary molecule OMS824 has received
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fast track designation from US FDA for Huntington’s disease. TAKꢀ063 (Takeda) and AMGꢀ
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79 (Amgen) are also presently in clinical trials.
Despite enormous efforts to develop a PDE10A inhibitor by several pharmaceutical companies
for more than a decade, so far no molecule has reached the market. This may be due to the
complexity involved in coꢀoptimizing and developing a drug for CNS indications such as
Schizophrenia or Huntington’s disease. There exists a need for developing and evaluating new
PDE10A inhibitors with improved efficacy and safety profile. The objective of this study was to
explore and generate a new chemical series of PDE10A inhibitors with good potency, selectivity
and efficacy in relevant animal models.
RESULTS AND DISCUSSION
Synthetic Chemistry. A diverse set of diaryl pyridine and pyridone derivatives required for our
study was prepared as shown in Schemes 1−7. A regioselective sequential double Suzuki
coupling strategy was used for the synthesis of the desired compounds. The coupling reaction of
5,6ꢀdibromopyridinꢀ2ꢀamine 1 with a suitable phenyl boronic acid in the presence of sodium
carbonate under Suzuki reaction conditions exclusively gave the 6ꢀarylꢀ5ꢀbromoꢀ2ꢀ
aminopyridine 2aꢀl (Scheme 1). The intermediates 2a−l were then subjected to a second Suzuki
coupling reaction with an appropriate arylꢀ or pyridyl boronic acid in the presence of cesium
15
carbonate to yield the diaryl aminopyridines 3a−l in good to excellent yield. Demethylation of
ethers 3a−k using hydrogen bromide in glacial acetic acid gave the corresponding phenols 4a−k.
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The benzyl ether 3l on hydrogenolysis using Pd/C gave 4l in 65% yields. The phenols 4a−l on
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coupling reaction with heterocylic halides or tosylates of the formula 5a−g afforded ethers 6a−x.
Reductive alkylation of 6a with formaldehyde in the presence of formic acid gave the
dimethylaminopyridine 7a. The compound 6b on Balz–Schiemann reaction using tertꢀbutyl
nitrite in the presence of tetrafluoroboric acid afforded the 2ꢀfluoropyridine derivative 7b in good
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yield.
Scheme 1. Synthesis of 2a−l, 3a−l, 4a−l, 6a−x and 7a−b
Reagents and conditions: (a) ArB(OH) , Pd(PPh ) , Na CO , MeOH, toluene, 110 °C, 16 h, 65−85%; (b)
2
3 4
2
3
4
ꢀMeꢀOPhB(OH) , Pd(PPh ) , Cs CO , dioxane, 100 °C, 16 h, 72−91%; (c) 33% HBr in glacial AcOH, 80
2
3 4
2
3
°
°
C, 14 h, 61−82%; (d) H , 10% Pd/C, EtOHꢀTHF (1:1), 50−60 psi, RT, 15 h, 65%; (e) Cs CO , DMF, 70
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2
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C, 48 h, 67−95%; (f) HCHO, HCOOH, reflux, 15 h, 51%; (g) (CH ) CONO, HBF , CHCl , RT, 18 h,
3 3
4
3
48%.
Analogous compounds bearing other functional groups such as an ester, carboxylic acid and a
hydroxymethyl group at the 2ꢀposition of the pyridine ring were prepared as shown in Scheme 2.
Thus, commercially available 5ꢀaminoꢀ6ꢀbromopyridineꢀ2ꢀcarbonitrile 8 on coupling reaction
with 4ꢀfluorophenylboronic acid under Suzuki reaction conditions afforded the corresponding
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aryl pyridine, which on diazotization with tertꢀbutyl nitrite followed by treatment with iodine
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gave the 5ꢀiodopicolinonitrile 9. The iodide 9 on Suzuki coupling reaction with 4ꢀ
hydroxyphenylboronic acid gave the phenol intermediate which on alkylation with 2ꢀ
(chloromethyl)quinoline 5a afforded compound 11. The intermediate 10 required for the
synthesis of compounds 12a−c was prepared from 9 in three steps. Thus, Suzuki coupling
reaction of 9 with 4ꢀhydroxyphenylboronic acid followed by base hydrolysis and esterification of
the resultant carboxylic acid yielded ester 10. The compound 10 was then coupled with 2ꢀ
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(chloromethyl)quinoline to give ester 12a. The ester 12a on alkaline hydrolysis afforded the
carboxylic acid 12b. Lithium aluminum hydride mediated reduction of the ester functionality in
12a afforded the hydroxylmethyl derivative 12c.
Scheme 2. Synthesis of compounds 11 and 12a−c
I
HO
H N
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a
c
N
CN
Br
N
CN
O
F
N
CO C H
2 2 5
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F
10
d e or f
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O
N
N
N
CN
N
R
F
F
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12a R = CO C H
2 2 5
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2b R = CO H
2
2c R = CH OH
2
Reagents and conditions: (a) (i) 4FꢀPhB(OH) , Pd(PPh ) , Na CO , MeOH, toluene, 110 °C, 16 h, 95%,
2
3 4
2
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(
ii) Iodine, (CH ) CONO, CHCl , RT, 1 h, 54%; (b) (i) 4ꢀHOꢀPhB(OH) , Pd(PPh ) , Cs CO , dioxane,
3 3 3 2 3 4 2 3
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00 °C, 16 h, 76%, (ii) 2ꢀ(chloromethyl)quinoline, K CO , DMF, 70 °C, 18 h, 69%; (c) (i) 4ꢀHOꢀ
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PhB(OH) , Pd(PPh ) , Cs CO , dioxane, 100 °C, overnight, 88%, (ii) 2N NaOH, EtOH, 1 h, RT, 81%,
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(iii) conc. H SO , EtOH, reflux, 16 h. 78%; (d) 2ꢀ(chloromethyl)quinoline, K CO , DMF, 40 °C, 18 h,
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73%; (e) 2N NaOH, EtOH, 14 h, RT, 89%; (f) LAH, THF, 0 °C, 30 min, 86%.
An approach developed for the synthesis of compounds bearing selected functional groups such
as nitro, amino, hydroxyl and fluorine at 3ꢀposition of the pyridine ring is shown in Scheme 3.
Thus, Suzuki coupling reaction of 3ꢀbromoꢀ5ꢀnitropyridinꢀ2ꢀol 13 with 4ꢀmethoxyphenylboronic
acid yielded 3ꢀarylꢀ5ꢀnitropyridinꢀ2ꢀol derivative which on treatment with POCl gave the 2ꢀ
3
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chloropyridine derivative 14. The intermediate 14 upon a second Suzuki coupling reaction with
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4ꢀ(fluorophenyl)boronic acid followed by hydrogen bromide assisted demethylation gave the
phenolic intermediate 15. Alkylation of 15 with 2ꢀ(chloromethyl)quinoline 5a gave compound
16a. Reduction of 16a with iron powder in the presence of aqueous ammonium chloride gave 3ꢀ
aminopyridine derivative 16b. Diazotization of 16b using sodium nitrite in the presence of
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sulfuric acid gave the targeted 3ꢀhydroxy pyridine derivative 17a. The 3ꢀfluoropyridine
derivative 17b was prepared by treating 16b with tertꢀbutyl nitrite in the presence of
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tetrafluoroboric acid as described in Scheme 1.
Scheme 3. Synthesis of pyridines 16a−b and 17a−b
Reagents and conditions: (a) (i) 4ꢀMeOꢀPhB(OH) , Pd(PPh ) , Cs CO , dioxane, 100 °C, 16 h, 61%, (ii)
2
3 4
2
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POCl , 120 °C, 2 h, 74%; (b) (i) 4FꢀPhB(OH) , Pd(PPh ) , Na CO , MeOH, toluene, 110 °C, 16 h, 88%,
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3 4
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(
ii) BBr , dichloromethane, 0 °C−RT, 14 h, 63%; (c) 2ꢀ(chloromethyl)quinoline, Cs CO , DMF, 70 °C, 48
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h, 72%; (d) Fe powder, aq. NH Cl, EtOH, RT, 30 min, 79%; (e) NaNO , aq. H SO , 0 °C, 10 min, 38%;
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(f) (CH ) CONO, HBF , CHCl , RT, 16 h, 29%.
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A diverse set of novel pyridones (pyridineꢀ2(1H)ꢀones) 18a−x was prepared by diazotization of
the corresponding 2ꢀaminopyridines 6a−x followed by in-situ hydrolysis of the corresponding
17
diazonium salts as shown in Scheme 4. The desired 2ꢀaminopyridine derivatives 6a−x with
pendent aryl or heteroaryl groups were prepared by lateꢀstage coupling of advanced
intermediates 4a−l with an assorted set of electrophile components 5a−g as described in Scheme
2
1. The details of Ar and R groups of 6a−x and 18a−x are given in Table 3 (vide infra). The
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targeted 6ꢀmethoxypyridine 19 and Nꢀmethylpyridone 20 required for structure activity
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relationship (SAR) analysis were prepared from pyridone 18b. Thus, pyridone 18b on alkylation
with methyl iodide in the presence of anhydrous potassium carbonate in DMF afforded an easily
separable mixture of 2ꢀmethoxypyridine 19 and Nꢀmethylpyridone 20.
Scheme 4. Synthesis of 18a−x, 19 and 20
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2
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R O
R O
a
Ar
N
NH2
O
Ar
N
H
O
O
6a-x
1
8a-x
O
N
N
b
1
8b
+
N
CH3
N
OCH3
F
F
1
9
20
Reagents and conditions: (a) NaNO , 2.0 M H SO , 0 °C−RT, 10 min, 75−85%, (b) CH I, K CO , DMF,
RT, 16 h (19: 44% and 20: 51%).
2
2
4
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2
3
The synthesis of targeted regioisomeric pyridones 22a−d bearing the alkoxyphenyl group on the
carbon adjacent to pyridine nitrogen was accomplished by reversing the Suzuki coupling
sequence as shown in Scheme 5. Thus, reaction of 5,6ꢀdibromopyridinꢀ2ꢀamine 1 with 4ꢀ
methoxyphenylboronic acid in the presence of sodium carbonate in a mixture of toluene and
methanol afforded the 2ꢀaryl pyridine, which was then subjected to the second Suzuki coupling
reaction with aryl boronic acid to yield the diaryl 2ꢀaminopyridines 21a−d. Demethylation of
21a−d using BBr in dichloromethane yielded the corresponding phenol intermediates 22a−d.
3
Alkylation of 22a−d with 2ꢀ(chloromethyl)quinoline 5a gave the ethers 23a−d. Finally, the
pyridones 24a−d were prepared by diazotization of amines 23a−d and in situ hydrolysis of the
corresponding diazonium salts.
Scheme 5. Synthesis of isomeric pyridones 24a−d
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Reagents and conditions: (a) (i) 4ꢀMeOꢀPhB(OH) , Pd(PPh ) , Na CO , MeOH, toluene, 110 °C, 16 h,
2
3 4
2
3
7
8−86%, (ii) ArB(OH) , Pd(PPh ) , Cs CO , dioxane, 90 °C, 16 h, 90−95%; (b) BBr , CH Cl , 0 °C−RT,
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3 4
2
3
3
2
2
1
4 h, 80−91%; (c) 2ꢀ(chloromethyl)quinoline, K CO , DMF, 70 °C, 16 h, 65−73%; (d) NaNO , H SO , 0
2
3
2
2
4
°
C, 10 min, 67−75%.
The prodrugs required for our study were prepared from selected pyridones 18a−b and 18f
Scheme 6). The reaction of pyridones 18a−b, and 18f with acetic anhydride in presence of
triethylamine afforded the corresponding pyridyl acetates 25a−c in good yields. The reaction of
8b with N,Nꢀdimethylcarbamoyl chloride in the presence of potassium carbonate in THF
(
1
afforded the carbamate 25d in moderate yield.
Scheme 6. Synthesis of prodrugs 25a−d
Reagents and conditions: (a) (CH CO) O, Et N, THF, RT, 16 h, 81–88%; (b) (Me) NCOCl, K CO , THF,
3
2
3
2
2
3
70 °C, 15 h, 71%.
Finally, we targeted the conversion of pyridone 18b to the corresponding Oꢀglucuronide 26 and
the synthetic approach developed has been delineated in Scheme 7. Thus, 18b on reaction with
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acetobromoꢀαꢀDꢀglucose using silver carbonate as a base smoothly afforded the corresponding
1
2
3
4
5
6
7
8
9
acetyl glucuronide which on hydrolysis under basic conditions gave glucuronide 26 in 80%
1
8
yield. To the best of our knowledge, there have been no literature reports on the formation of
glucuronide conjugates of pyridones via its enol tautomer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 7. Synthesis of Oꢀglucuronide 26
Reagents and conditions: (a) acetobromoꢀαꢀDꢀglucose, AgCO , RT, 3 days; (b) 1.0 M NaOH, acetone,
RT, 1.0 h.
3
Structure activity relationship (SAR) analysis. Several structurally diverse molecules have
been reported in the literature as PDE10A inhibitors. In an effort to discover new chemotypes,
we compared structural and topographical similarities of a large number of molecules from our
internal compound library with known PDE10A inhibitors reported in the literature. A number of
molecules were selected for screening from our internal library based on structure and shape
similarity, without considering their atom properties. Selected compounds 3a−c were of
particular interest to us due to their structural similarity with known PDE10A inhibitors reported
11
by Pfizer. These compounds were then screened against hPDE10A enzyme. The inhibitory
3
activity of 3a−c was measured using a scintillation proximity assay with [ H]ꢀcAMP as the
substrate by measuring hydrolysis of cAMP to AMP using recombinant human PDE10A enzyme
1
9
at 1.0 and 10 µM concentrations. Unfortunately, these compounds did not show any activity at
both the concentrations. We presumed that lack of activity of these compounds may be due to the
absence of the critical binding interaction of these molecules with PDE10A enzyme. We
hypothesized that replacing the methyl group in 3a−c with a quinolinemethyl group may result in
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11b
Page 10 of 61
the critical binding interaction with Tyrꢀ683 of PDE10A enzyme. Thus, the quinolinemethyl
ether derivative 6a was prepared and screened against hPDE10A. As expected, 6a showed
significant inhibition of the enzyme at 1.0 and 10.0 µM concentrations of the compound (Table
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
). It showed an IC of 2435.5 nM. The 5,6ꢀdiphenylꢀ2ꢀaminopyridine derivatives 6b and 6c,
50
prepared around the hit compound 6a also showed similar binding affinity to PDE10A (Table 1).
The fluoro derivative 6b showed similar results while the corresponding chloro derivative 6c
showed significant improvement in potency (364.2 nM). The amino group in 6a was then
replaced by N,Nꢀdimethylamino group as in the case of 7a, which resulted in complete loss of
binding affinity with the PDE10A enzyme. In another modification, the amino group in 6b was
replaced by a fluorine atom as in the case of 7b to give a potency of 350.9 nM, which was
comparable to the potency of 6c.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. SAR of pyridines 3a−c, 6a−c and 7a−b
3
a,b
Compd
R
Ar
R
% Inhib
0.3/1
2/3
hIC (nM)
5
0
c
3
3
a
Me
Me
Me
Ph
NH
NH
2
2
2
2
ND
ND
ND
b
4FꢀPh
3c
4ClꢀPh NH
0.2/3
33/79
38/72
71/83
7/12
d
6
6
6
a
b
c
Qu
Qu
Qu
Qu
Qu
Ph
NH
NH
2435.5 ± 86.2
2740.1 ± 98.5
364.2 ± 22.1
ND
4FꢀPh
2
2
4ClꢀPh NH
7a
7b
Ph
N(Me)
F
3
4FꢀPh
62/73
350.9 ± 29.5
a
b
Percentage inhibition is measured at 1.0 and 10.0 µM concentrations of the compounds. Values are
c
d
mean of two independent experiments in duplicate. ND refers to ‘not determined’. Qu represents
quinolinꢀ2ꢀylmethyl group.
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Pyridine derivatives with cyano, ester, carboxylic acid and hydroxymethyl groups at 2ꢀposition
1
2
3
4
5
6
7
8
9
of the pyridine ring were also studied and the results are shown in Table 2. The 2ꢀcyanopyridine
11 showed poor enzyme inhibitory activity at 1.0 and 10 µM concentrations of the compound.
The ester derivative 12a and carboxylic acid 12b also showed poor binding affinity. The
hydroxymethyl derivative 12c displayed a moderate potency of 120.1 nM. These results
demonstrate that all the four functional groups at 2ꢀposition of the pyridine ring failed to give
favourable binding interactions with the enzyme. With limited success in achieving good
potency with 11 and 12a−c, we profiled selected compounds with functional groups at 3ꢀposition
of the pyridine ring. The novel 5,6ꢀdiarylpyridines bearing a nitro, amino, hydroxyl or a fluorine
substitution at the 3ꢀposition of the pyridine ring were screened. The nitro pyridine 16a showed
poor inhibitory activity at 1.0 and 10 µM concentrations of the compound. The corresponding
aminopyridine derivative 16b showed a slightly better profile. A hydroxyl group or a fluorine
atom at the 3ꢀposition of the pyridine ring as in the case of 17a and 17b resulted in moderate
potency of 226.6 nM and 193.6 nM respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. SAR of compounds 11, 12a−c, 16a−b and 17a−b
1
a,b
c
Compd
R
R
% Inhib
12 / 23
3 / 39
hIC (nM)
5
0
d
1
1
1
CN
CO
CO
CH
H
H
H
H
ND
ND
ND
2a
2
2
2
Et
H
12b
47 / 87
76 / 95
7 / 23
1
1
1
2c
6a
6b
OH H
NO
120.1 ± 9.7
ND
2
H
NH
51 / 94
ND
2
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1
7a
7b
H
H
OH
F
79 / 92
226.6 ± 15.1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
80 / 83
193.6 ± 16.2
a
b
Percentage inhibition is measured at 1.0 and 10.0 µM concentrations of the compounds. Percentage
inhibition are mean of two independent experiments in duplicate. IC values are mean ± SD of two
independent experiments using 7 to 9ꢀpoint concentrationꢀresponse curve. ND refers to ‘not determined’.
c
5
0
d
With limited success in achieving good potency in case of pyridine derivatives 6a−c and 7a−b,
we further planned to evaluate the corresponding pyridineꢀ2(1H)ꢀones. We were pleased to see
that the conversion of Hꢀbond donor amino group in 6a with the Hꢀbond acceptor carbonyl group
as in the case of pyridone 18a resulted in complete inhibition of the enzyme at 1.0 and 10.0 µM
concentrations of the compound. It showed 2200 fold improvement in potency with IC value of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
1
.12 nM (Table 3). We then evaluated the in vitro potency of pyridone analogues with several
quinoline ring replacement groups to derive reliable SAR for this series. Concurrent changes
were also carried out with selected aryl group replacements at 6ꢀposition of the pyridone ring.
The fluoro and chloro derivatives 18b and 18c also showed single digit nanomolar potency. The
regioisomeric fluoro phenyl derivatives 18d and 18e also showed comparable potencies. The
difluoro derivative 18f also retained potency. While both 18g and 18h showed single digit
nanomolar potency, the 4ꢀmethyl derivative 18h was twice as potent as the 2ꢀmethyl derivative
18g. The pyridone derivative with an electron withdrawing trifluoromethyl group at 4ꢀposition of
the phenyl ring as in the case of 18i resulted in significant loss of potency. The presence of
electron donating methoxy group in 18j resulted in retention of potency. Among the isomeric
pyridine derivatives 18k and 18l, the 4ꢀpyridyl derivative 18l was found to be 10 fold more
potent than the corresponding 3ꢀpyridyl derivative 18k. A topologically similar naphthyl
derivative 18m, which is devoid of the desired ring nitrogen resulted in complete loss of potency
(
3.5 and 5.8% at 1.0 and 10.0 µM concentration of the compound). Compounds 18n and 18o
bearing 1,5ꢀnaphthyridine ring, wherein an additional ring nitrogen is present, showed loss of
potency compared to the corresponding quinoline analogues 18a−b. The compound 18p,
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Journal of Medicinal Chemistry
wherein the quinoline is replaced with a monocyclic pyridine ring resulted in 124 fold loss in
1
2
3
4
5
6
7
8
9
potency, despite bearing suitably positioned nitrogen atom. Replacement of quinoline ring with
1,3ꢀbenzothiazol as in the case of 18q resulted in around 570 fold loss in potency. The pyridines
18r−u bearing 1Hꢀpyrrolo[3,2ꢀb]pyridine as the bicyclic core displayed subꢀnanomolar potency.
The 1ꢀmethylꢀ1Hꢀpyrrolo[3,2ꢀb]pyridine 18v−x, that are devoid of NH donor group also retained
good potency except in the case of 18x.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. SAR of 18a−x and 19 and 20
2
a
Compd
Ar
R
hIC (nM)
50
1
1
1
1
8a
8b
8c
8d
1.12 ± 0.09
1.62 ± 0.07
1.11 ± 0.05
2.71 ± 0.12
1
1
8e
8f
1.23 ± 0.07
1.41± 0.11
1
8g
5.02 ± 0.52
1
1
1
1
1
8h
8i
1.85 ± 0.12
6.46 ± 0.42
1.90 ± 0.23
18.62 ± 1.03
1.22 ± 0.10
8j
8k
8l
b
1
1
8m
8n
ND
21.20 ± 1.01
21.69 ± 1.15
136.6 ± 8.36
1
1
8o
8p
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1
8q
8r
926.4 ± 60.2
1
0.21 ± 0.01
0.33 ± 0.03
0.30 ± 0.02
0.54 ± 0.05
1.73 ± 0.10
0.73 ± 0.07
11.96 ± 0.65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
1
8s
8t
8u
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18v
1
1
8w
8x
c
19
ꢀ
ꢀ
ꢀ
ꢀ
˃ 10000
2
0
2984.5 ± 192.2
a
IC values are mean ± SD of two independent experiments using 7 to 9ꢀpoint concentrationꢀresponse
curve. ND refers to IC value was not determined, showed 3% and 6% inhibition at 1.0 and 10.0 µM
concentrations, respectively. Compound 19 showed an inhibition of 21 and 28% at 1.0 and 10 µM
5
0
b
5
0
c
concentrations, respectively.
The 2ꢀmethoxypyridine 19 and the isomeric Nꢀmethylpyridone 20 were of special interest to us
to understand the basis of high potency of most of the pyridones studied (Scheme 4 and Table 3)
as compound 19 is devoid of the carbonyl functionality while compound 20 has locked or nonꢀ
enolizable carbonyl functionality. Compound 19 showed nearly complete loss of potency (21%
and 28% inhibition at 1.0 and 10 µM concentrations respectively). Compound 20 showed an IC50
value of 2984.5 nM, which is approximately 1840 fold reduction in potency relative to the parent
pyridone 18b. The loss of potency in 19 and 20 suggests the importance of having both
enolizable carbonyl group and a free NH group to achieve good potency for this class of
PDE10A inhibitors.
We then evaluated the binding affinity and potency of regioisomeric pyridones 24a−d and the
results are shown in Table 4. It was observed that transposition of the aryl groups at 5ꢀ and 6ꢀ
position of the pyridone ring resulted consistent and significant reduction in potency. All the
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Journal of Medicinal Chemistry
compounds studied (24a−d) were less potent than the corresponding regioisomers 18a−b, 18i
1
2
3
4
5
6
7
8
9
and 18l. For example, 24a is approximately 25 times less potent than the corresponding
regioisomer 18a. Similar trends were observed in the case of 24b and 24c, whereas the effect
was less pronounced in the case of 24d.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. SAR of isomeric pyridones 24a−d
a
Compd
Ar
hIC (nM)
50
24a
28.9 ± 2.1
41.5 ± 4.7
37.0 ± 3.3
3.3 ± 0.24
2
2
4b
4c
24d
a
IC values are mean ± SD of two independent experiments using 7 to 9ꢀpoint concentrationꢀresponse
50
curve.
The detailed SAR analysis discussed in Tables 1−4 clearly shows that pyridone derivatives, in
general, were superior in potency compared to the related pyridine derivatives studied.
Therefore, as a part of the lead optimization strategy, selected pyridones from Table 3 were
screened against a broader panel of PDEs to assess the selectivity of this class of compounds and
the results are given in Table 5. The PDE selectivity assay of compounds 18a−c, 18f, 18l, 18r,
18t and 18v−w was carried out using human recombinant PDEs 1−5, 7−9 and 11 at 1.0 and 10.0
1
9
µM concentrations of the compounds. The in vitro radiometric assay results show that these
compounds are at least 5800ꢀfold more selective towards PDE10A enzyme over other related
PDEs.
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Table 5. PDE selectivity of compounds 18a−c, 18f, 18l, 18r, 8t and 18v−w
a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Percentage inhibition at 1.0 and 10.0 µM concentrations
Compd
8a
18b
hPDE1 hPDE2 hPDE3
3 / 10 15 / 30 3 / 5
hPDE4 hPDE5 hPDE7 hPDE8 hPDE9
hPDE11
13 / 19
1
2 / 14
3 / 36
29/ 35
3 / 8
12 / 6
0 / 0
7 / 8
2 / 8
3 / 0
0 / 0
9 / 8
0 /0
1 / 1
20 / 27 10 / 26 0.7 / 7
11 / 26 12 / 34 1 / 3
13 / 48 15 / 21 7/ 31
0 / 0
15 / 14
1 / 13
7 / 9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
8c
8f
8l
0 / 9
0 / 2
0 / 15
ND
8 / 25
2 / 12
3 / 34
8 / 7
14/ 36
22/ 50
9/ 40
12 / 18
8 / 5
12 / 18 11 / 12 3 / 4
b
ND
16 / 43 2 / 9
0.3 / 0
29 / 30 16 / 18 1 / 0.8
10 / 13
0 / 13
19 / 22
11 / 1
16 / 16
7 / 15
0 /0
8r
11 / 21 9 / 14
12/ 17
18t
8v
18w
ND
39 / 47 32/ 29
6 / 7 0/ 8
24 / 29 30/ 39
0 / 0
1
40 / 35 11 / 10 0.5 / 0.9 ND
29 / 31 32 / 37 9 / 16 ND
11 / 34
0 / 0
a
Percentage inhibition (1.0/10 µM) values are mean of two independent experiments run in duplicate for
b
both concentrations. ND refers to ‘not determined’.
In vitro liver microsomal stability of selected compounds
The above potent pyridones were subjected to in vitro metabolic stability studies using pooled
liver microsomal preparations of wistar rat, CD1 mouse, beagle dog, cynomolgus monkey and
human. The metabolic stability results are shown in Table 6. Briefly, the compounds were
incubated at a final concentration of 1.0 µM with 1.0 mg/mL of microsomal protein fortified
with NADPH (2.4 mM) at 37 °C for 60 min. The in vitro metabolic stability of compounds was
determined by measuring their disappearance using LCꢀMS/MS. Compound 18a showed
moderate stability in the rat, dog and human while it showed poor stability in mouse and monkey
liver microsomes. The metabolic stability profile of 4ꢀsubstituted derivatives 18b and 18c was
substantially better in mouse and monkey liver microsomes compared to the corresponding
unsubstituted phenyl derivative 18a. The most potent pyridones 18r and 18s bearing the 1Hꢀ
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Journal of Medicinal Chemistry
pyrrolo[3,2ꢀb]pyridine side chain showed poor stability across the species except in the dog
1
2
3
4
5
6
7
8
9
suggesting extensive metabolism of this ring. Similarly, the corresponding Nꢀmethylpyrrolo[3,2ꢀ
b]pyridine derivative 18v also showed poor metabolic stability in all species except the dog. The
2,4ꢀdifluoroderivative 18f showed good stability in mouse, rat, dog and human but moderate
stability in monkey. The 4ꢀmethyl derivative 18h showed good metabolic stability in the dog and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
human, but poor stability in rat, mouse and monkey liver microsomes. The pyridine derivative
18l showed good metabolic stability in all species studied. Thus, in general, compounds bearing
quinolinylꢀ2ꢀylmethyl side chain were more stable than the more potent analogues bearing 1Hꢀ
pyrrolo[3,2ꢀb]pyridine moiety.
a,b
Table 6. Metabolic stability data of 18a−c, 18f, 18h, 18l, 18r−s, 18v
Compd Rat
8a
18b
Mouse Dog
Monkey Human
1
59.7
48.1
60.6
75.6
18.5
67.1
11.1
15.5
4.3
3.5
70.0
99.0
3.0
62.6
74.0
92.2
66.4
65.4
77.7
1.0
54.0
60.9
77.0
41.9
72.0
20.3
0.8
49.0
1
1
1
8c
100.0 53.8
8f
98.3
88.7
91.1
42.0
42.1
35.0
29.0
72.5
3.6
8h
18l
1
8r
8s
1
6.6
0.6
18v
3.9
51.7 b 1.3
4.4
a
Values are mean of three replicates. Percentage remaining at 60 min in liver microsomal preparations.
20
Pharmacokinetics and in vivo metabolism of 18a−b, 18f and the prodrugs 25a−d
After evaluation of potency, selectivity and in vitro metabolic stability data of several
compounds, we decided to assess the in vivo pharmacokinetic (PK) profile of selected
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compounds 18a−b and 18f. A single dose (10 mg/kg) oral PK study of 18a−b and 18f was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
initially performed in male Sprague Dawley (SD) rats and the PK parameters are shown in Table
7. Compounds 18a−b and 18f exhibited very poor pharmacokinetics, with Cmax (9 − 73 ng/mL)
and AUC0ꢀt (38 − 370 ng.h/mL) upon oral dosing as a methylcellulose suspension. The poor
systemic exposures of these compounds did not correlate with the moderate to high in vitro
metabolic stability as shown in Table 7. It may be noted that these compounds have favourable
physicochemical properties (Mol. Wt.: 404−440; cLogP: 4.46−4.77; tPSA: ~ 50.6) amenable for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
oral absorption. In a parallel artificial membrane permeability assay (PAMPA), these
ꢀ6
compounds were found to be highly permeable (P : 2.9 − 3.6 x 10 cm/s). These
app
physicochemical properties also appear to be favorable for crossing the blood brain barrier by
passive diffusion. However, compounds 18a−b and 18f showed poor aqueous solubility (< 0.1
µg/mL) which may have been due to the high melting nature of these pyridone compounds
(226−246 °C). To address the poor solubility profile of these compounds, we sought to prepare
corresponding prodrugs 25a−d (Scheme 6). Prodrugs 25a−d exhibited favorable melting points
(143−167 °C) but resulted in no improvement in aqueous solubility. However, unlike the
pyridones, these compounds showed very good solubility in organic solvents.
Although the prodrug approach did not improve aqueous solubility of these compounds, we
decided to study the oral PK profile of 25a−d in SD rats. Following oral administration of
acetate prodrugs, quantifiable levels of parent pyridones were found in the plasma samples up to
8
h post dose in all the animals (n = 3) studied. The prodrug levels were not detected in the
plasma samples at any time points. The acetate prodrugs 25a−c showed improved PK profile
upon oral dosing at 11.04 mg/kg (equivalent to 10 mg/kg of the parent compounds) in SD rats as
shown in Table 7. The carbamate prodrug 25d failed to improve the PK profile. Among the
prodrugs studied, the best PK profile was observed for 25b. It showed a mean Cmax of 248 ± 68
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Journal of Medicinal Chemistry
ng/mL and AUC0ꢀt of 2436 ± 390 ng.h/mL. Thus, the strategy to dose 18b as its prodrug 25b
1
2
3
4
5
6
7
8
9
substantially improved the PK profile of the parent compound. Further, the brain to plasma ratio
was determined for compounds 18a−b, 18f at 1 h postꢀdose. Compound 18a and 18b showed
good brain to plasma ratio of 1.5 and 1.1 respectively, while 18f showed a relatively poor value
of 0.5. Based on the superior PK profile and other favourable data discussed above, pyridone 18b
as its acetate prodrug 25b was selected as a lead compound for further profiling.
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11
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13
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18
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23
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25
26
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60
a
b
Table 7. PK data of 18a−b, 18f when doses as such and as their prodrugs 25a−d in SD rats
c
Drug / Prodrug Cmax (ng/mL)
18a / 25a 9 ± 3 / 372 ± 58
Tmax (h)
AUC (ng.h/mL)
B/P ratio
1.5
0ꢀt
0.5 / 1.0
38 ± 20 / 1497 ± 615
359 ± 272 / 2436 ± 1025
370 ± 264 / 1764 ± 226
359 ± 272 / 182 ± 44
1
1
1
8b / 25b
8f / 25c
8b / 25d
44 ± 12 / 372 ± 128 0.5 / 1.0
1.1
73 ± 57 / 266 ± 50
4.0 / 2.0
0.5
d
44 ± 12 / 42 ± 19
0.5 / 1.0
ND
a
b
.
Mean ± SD values (n = 3). 11 04 mg/kg dose of the prodrug is equivalent to 10 mg/kg of the parent.
c
d
Tmax is median. ND refers to ‘not determined’.
The parent compound 18b was also evaluated on selected panel of receptors, ion channels,
transporters and enzymes (CEREP) at a concentration of 10 ꢁM. In the binding assay
experiments, compound 18b showed no significant effect on a majority of the enzymes in the
panel. It showed an IC of 6.7 nM on h5HT2b receptor binding assay. The compound showed
5
0
relatively low binding affinity towards norepinephrine transporter, LTD4 receptor and dopamine
receptor with IC values above 1200 nM. The followꢀup studies were conducted for h5HT2b
50
receptor to assess its functional modulation. Compound 18b showed functional antagonism on
h5HT2b receptor with an IC50 of 220 nM and therefore is 135ꢀfold more selective towards
PDE10A. Compound 18b was then tested for its hERG potassium channel inhibition to evaluate
its cardiovascular safety. It showed 42.4% inhibition at 10 µM concentration in hERG302ꢀHEK
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Page 20 of 61
cells in a patch clamp electrophysiology assay and was classified as a low potency hERG
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1
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1
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2
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6
channel blocker.
Characterization and quantification of phase 2 metabolite of pyridone 18b
We proposed that the low exposure of 18b is due to the Oꢀglucuronide conjugate formation
through pyridineꢀ2ꢀol tautomer. As discussed in the chemistry section, the pure (96% by HPLC)
Oꢀglucuronide 26 of pyridone 18b was prepared and characterized to compare with the
metabolites isolated from the in vitro liver and intestinal microsomal incubations. The in vitro
microsomal incubations of 18b were carried out in the presence of uridine 5'ꢀdiphosphoꢀ
glucuronic acid (UDPGA) as cofactor. The phase 2 metabolite identification studies were carried
on LCꢀMS/MS using a neutral loss (loss of 176 amu in positive ionization mode) scan. We
observed that 18b underwent Oꢀglucuronidation in these incubations and it was confirmed by
comparison of the metabolite with synthetic Oꢀglucuronide 26. Details of the experiment and
characterization studies are given in the supporting information.
0
1
2
3
4
5
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7
8
9
0
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3
4
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6
7
8
9
0
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0
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0
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7
8
9
0
Additional in vivo studies were carried out for quantification of glucuronide metabolite 26 by
dosing 18b as its acetate prodrug 25b. Plasma level of glucuronide conjugate was quantified
using LCꢀMS/MS analysis. Prodrug 25b was administered both orally (0.5% MC suspension)
and intravenously (20% NMP + 20% ethanol + 10% PG + 50% premix of 3:2 of PEG 200 and
water) to SD rat, beagle dog and cynomolgus monkey. As shown in Table 8, the percentage
exposure of glucuronide metabolite to parent 18b was approximately 3−9 times lower upon
intravenous (i.v.) administration as compared to oral administration in all the three species
studied. Systemic exposure of the glucuronide was found to be in the order monkey > dog > rat,
upon oral administration of 25b. These investigations revealed that poor oral PK profile of 18b
and other pyridone derivatives may partly be attributed to the significant first pass Oꢀ
glucuronidation in liver as well as in intestine.
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Journal of Medicinal Chemistry
Table 8: PK data of 18b after i.v. and oral administration of 25b
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2
3
4
5
6
7
8
9
a
b
Species
Rat
Route
Dose
Cmax
AUC0ꢀ24
CL
Tmax 26/18b
(
mg/Kg) (ng/mL) (ng.h/mL) (mL/min/kg)
(h)
ratio
0.34
1.33
0.25
2.19
1.32
4.06
c
d
i.v.
1.10
ND
372
ND
49
492
28
NA
oral
i.v.
11.04
1.10
2436
841
ND
20
1.0
NA
1.0
NA
6.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dog
oral
i.v.
11.04
1.10
555
ND
13
ND
38
1314
345
Monkey
oral
11.04
ND
a
Doses 1.10 mg/kg and 11.04 mg/kg of 25b are equivalent to 1.0 and 10 mg/kg doses of the parent
b
c
d
compound 18b, respectively. AUC ratio of 26 to 18b. ND refers to ‘not determined’. NA refers to ‘not
applicable’.
In vivo pharmacology:
Having achieved good potency, selectivity and acceptable pharmacokinetics and drug
metabolism (PKDM) profile for 18b, we decided to study this compound in relevant rodent
models of psychosis. The calculated physicochemical properties of 18b (Mol. Wt.: 422; cLogP:
4.6; tPSA: 50.6) appeared to be favourable to achieve good brain levels and efficacy in animal
models. To establish a reliable correlation between in vitro potency, pharmacokinetics (PK) and
pharmacodynamics (PD), compound 18b was also screened against the rat and mouse PDE10A
enzyme. It showed an IC of 4.3 nM in rat and 2.6 nM in mouse, which is close to the human
50
potency of 1.6 nM. As discussed above, 18b is poorly bioavailable and therefore its acetate
prodrug 25b was selected for in vivo dosing. The compound was tested in rat and mouse models
that are predictive of antiꢀpsychotic activity in humans. The details of the studies are given in Fig
1, 2 and 3.
Firstly, compound 25b was evaluated in apomorphineꢀinduced compulsive climbing and
sniffing behavior in male CD1 mice, a model constructed on dopaminergic hypothesis of
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2
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Page 22 of 61
schizophrenia (Fig. 1). Apomorphine (1.0 mg/kg, s.c.) treated vehicle control group exhibited
robust climbing and sniffing behavior with cumulative scores of 43.1 ± 3.6 and 11.8 ± 0.6,
respectively. The compound 25b upon intraperitoneal administration at 0.3, 1, 3, 10, and 30
mg/kg produced doseꢀdependent inhibition of apomorphine induced climbing and sniffing
behaviors with ED values of 3.76 mg/kg and 18.75 mg/kg respectively. These results show that
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
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1
1
2
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2
2
2
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5
0
0
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0
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2
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0
1
2
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0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
the PDE10A inhibitor 25b has an antipsychotic profile similar to a D receptor antagonist in
2
alleviating dopamine related dysfunction in psychosis. An ED ratio of around 5 for sniffing vs.
5
0
climbing demonstrates selectivity of compound 25b for limbic over striatal brain region, which
predicts atypical antiꢀpsychotic profile of this compound.
1
20
Climbing
Sniffing
100
80
60
40
20
0
0.3
1
3
10
30
-20
Compound 25b (mg/kg)
Figure 1. Effect of compound 25b on apomorphineꢀinduced climbing and sniffing.
Secondly, the compound 25b was evaluated in MKꢀ801 (dizocilpine)ꢀinduced psychosis model
2
3
in female SD rats, a model constructed on glutamatergic hypothesis of schizophrenia.
Dizocilpine, a NꢀmethylꢀDꢀaspartate (NMDA) receptor antagonist, induces both positive and
negative symptoms of schizophrenia. In this model, animals were dosed with dizocilpine at 0.2
mg/kg intraperitoneally, and were scored for behavioral signs related to psychosis i.e. hyperꢀ
locomotion, stereotypy and ataxia. The compound 25b was administered orally 1 h prior to
dizocilpine injection at 3, 10 and 15 mg/kg dose. The compound 25b showed reversal of
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Journal of Medicinal Chemistry
dizocilpineꢀinduced hyperꢀlocomotion, stereotypy and ataxia in a doseꢀdependent manner with
1
2
3
4
5
6
7
8
9
ED50 values of 7.94, 20.29 and 8.75 mg/kg, respectively (Fig. 2). The positive comparator,
risperidone, used in this model also produced excellent efficacy. As shown in Figure 3, around 3ꢀ
fold higher ED value for stereotypic behavior over hyperꢀlocomotion and ataxia was observed,
5
0
which is suggestive of an atypical antipsychoticꢀlike profile of compound 25b. Data from this
model clearly demonstrate the ability of PDE10A inhibitor to alleviate the glutamatergic
dysfunction of psychosis.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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60
6
5
0
0
Sham
Vehicle
Risperidone 1 mg/kg, i.p
Compound 25b 3 mg/kg, p.o.
Compound 25b 10 mg/kg, p.o.
Compound 25b 15 mg/kg, p.o.
21%
*
**
40
5
3%
3
2
1
0
0
0
0
***
74%
*
**
82%
4
9%
*
**
32%
***
1
6% 43%
78%
**
6
8%
*
**
89%
*
**
***
***
Locomotion
Stereotypy
Ataxia
Figure 2. Effect of 25b on MKꢀ801ꢀinduced psychotic behavior in female SD rats. Percentage inhibition
depicted over each treatment bar. ** p <0.01; *** p <0.001 vs. vehicle; Oneꢀway ANOVA / Tukey’s test.
Finally, we tested compound 25b in quipazine (5ꢀHT agonist)ꢀinduced head twitch in male SD
2
A
24
rats, a model constructed on serotonergic hypothesis of schizophrenia. Compound 25b was
administered orally at 3, 10, 30 and 60 mg/kg, 1.0 h, before the quipazine injection (1 mg/kg,
s.c.). In vehicle treated animals, quipazine induced the head twitch behavior, which was dose
dependently inhibited by compound 25b with an ED of 16.3 mg/kg (Figure 3). Risperidone, a
5
0
positive comparator used in the experiment also exhibited excellent inhibition of quipazine
induced behavior in this model. The data from this experiment suggests that PDE10A inhibitor
can also improve the serotonergic dysfunction of psychosis.
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Sham
Vehicle
Risperidone 0.1 mg/kg, i.p.
Compound 25b 3 mg/kg, p.o.
Compound 25b 10 mg/kg, p.o.
Compound 25b 30 mg/kg, p.o.
Compound 25b 60 mg/kg, p.o.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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2
2
2
2
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5
5
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7
6
5
0
0
0
40
28%
4
0%
3
0
0
*
57%
8
3%
**
2
78%
*
**
*
**
0
1
2
3
4
5
6
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8
9
0
1
2
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0
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0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
0
Quipazine (1 mg/kg, s.c.)
Figure 3. Effect of compound 25b on quipazineꢀinduced head twitches in male SD rats. Percentage
inhibition depicted over each treatment bar. * p < 0.05, ** p <0.01; *** p <0.001 vs. vehicle; Oneꢀway
ANOVA / Tukey’s test.
In summary, a series of potent and highly selective novel biaryl pyridone PDE10A inhibitors
were discovered through focused library screening and hit to lead optimization through extensive
analogue synthesis. Several potent compounds from this class showed very good selectivity over
other related PDEs and good in vitro microsomal stability across species. This novel class of
compounds was screened for PDE10A potency, selectivity, physicochemical properties, CNS
penetration and in vivo efficacy to yield 18b. The selected compound 18b however showed poor
pharmacokinetic properties upon oral administration. Careful metabolite profiling revealed that
the formation of glucuronide conjugate 26 in both liver and intestine is responsible for relatively
low systemic exposure. The issue of poor PKDM profile was addressed by converting the
pyridone 18b to the corresponding enol acetate prodrug 25b. The prodrug 25b upon oral dosing
quantitatively released the parent compound and showed a 6ꢀfold increase in the exposure. The
excellent in vivo efficacy results in selected antipsychotic rodent models give scope for further
pharmacological and toxicological evaluation of this compound.
EXPERIMENTAL SECTION
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Journal of Medicinal Chemistry
General chemistry. Melting points are uncorrected. Infrared spectra were recorded on Perkin
1
13
1
2
3
4
5
6
7
8
9
Elmer Spectrum One FTꢀIR Spectrometer. H and C NMR spectra were recorded on a Varian
3
00 MHz FT NMR spectrometer in either CDCl3 or DMSOꢀd6 as specified using
tetramethylsilane as internal standard. Chemical shifts are quoted in ppm (δ) relative to TMS
1
( H) using residual protonated solvent as internal standard. Routine mass spectra (MS) were
10
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17
18
19
20
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22
23
24
25
26
27
28
29
30
31
32
33
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35
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
recorded on a Thermo Finnigan model LCQ Deca XP Max spectrometer using ESI or APCI
source at positive or negative polarity mode by direct infusion method. Highꢀresolution mass
spectra (HRMS) of test compounds were measured on a Thermo Scientific LTQ Orbitrap
Discovery MS system coupled with a LQT Tune Plus software (version 2.5.5 SPI) operating in a
positive electron spray ionization (ESI) mode. The chemical purity of the test compounds was
determined by reversed phase HPLC on a Shimadzu or Waters system. The analyses were
performed on Inertsil ODSꢀ3 (100 x 4.6 mm i.d., 3 µm) column using a binary gradient elution
program. The solvents were methanol or acetonitrile as organic phase and 0.1% trifluoroacetic
acid (TFA) in water as aqueous phase. A PDA detector was used and the UV detection
wavelengths for the compounds were selected according to their characteristic absorption
maxima. The injection volume was 20 µL (~ 500 ppm) and the flow rate was 0.8 mL/min. All
test compounds used in biological assay were greater than 95% pure by this method.
1
5a
5ꢀBromoꢀ6ꢀphenylpyridinꢀ2ꢀamine (2a); Typical procedure. To a stirred solution of 5,6ꢀ
dibromopyridinꢀ2ꢀamine (10.0 g, 42.21 mmol) in a mixture of methanol (20.0 mL) and toluene
(
200 mL) was added phenylboronic acid (5.15 g, 42.23 mmol) followed by sodium carbonate
8.95 g, 84.44 mmol) dissolved in water (50 mL). The mixture was purged with nitrogen for 15
(
min. Tetrakis(triphenylphosphine)palladium(0) (1.46 g, 1.26 mmol) was added to the mixture
and heated at 110 °C overnight. The reaction mixture was cooled to room temperature and
diluted with water (500 mL). The mixture was extracted with ethyl acetate (3 x 250 mL). The
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combined organic layers were washed with water (2 x 250 mL) and dried over anhydrous
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Na SO . The solvent was distilled off under reduced pressure and the crude product thus
2
4
obtained was purified by flash silica gel column chromatography using 15% ethyl acetate in
petroleum ether to give 7.21 g (68.6%) of the product as a pale yellow solid.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Intermediates 2b−l were prepared as described in the typical procedure for 2a by coupling 5,6ꢀ
dibromopyridinꢀ2ꢀamine and appropriate arylꢀ or pyridylboronic acid under Suzuki reaction
conditions. The characterization data for 2a−l is given in the supporting information.
5ꢀ(4ꢀMethoxyphenyl)ꢀ6ꢀphenylpyridinꢀ2ꢀamine (3a); Typical procedure. To a stirred solution
of 5ꢀbromoꢀ6ꢀphenylpyridinꢀ2ꢀamine 2a (7.0 g, 35.58 mmol) in dioxane (80 mL) were added 4ꢀ
methoxyphenylboronic acid (5.40 g, 35.53 mmol), and cesium carbonate (23.18 g, 71.14 mmol)
dissolved in water (40 mL). The mixture was purged with nitrogen for 15 min.
Tetrakis(triphenylphosphine)palladium(0) (1.22 g, 1.05 mmol) was added to the mixture and
heated overnight at 90 °C under nitrogen atmosphere. The reaction mixture was cooled and
diluted with water (300 mL). The mixture was extracted with ethyl acetate (3 x 200 mL). The
combined organic extracts were washed with water (2 x 250 mL) and dried over anhydrous
Na SO . The solvent was evaporated under reduced pressure to result a brown viscous residue.
2
4
The residue was purified by flash silica gel column chromatography using 20% ethyl acetate in
1
petroleum ether to afford 5.91 g (76%) of the product as an offꢀwhite solid. Mp 110−112 °C; H
NMR (300 MHz, DMSOꢀd ) δ 3.79 (s, 3H), 6.03 (br s, 2H), 6.49 (d, J = 8.4 Hz, 1H), 6.77 (d, J =
6
8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 7.21 (s, 5H), 7.38 (d, J = 8.1 Hz, 1H); APCIꢀMS (m/z) 277
+
(
M+H) .
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Journal of Medicinal Chemistry
Intermediates 3b−l were prepared as described above by coupling 2b−l with 4ꢀ
1
2
3
4
5
6
7
8
9
methoxyphenylboronic acid under Suzuki reaction conditions. The spectral data of these
intermediates is given in the supporting information.
4ꢀ(6ꢀAminoꢀ2ꢀphenylpyridinꢀ3ꢀyl)phenol (4a). Hydrobromic acid in glacial acetic acid (33 %
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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49
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60
(
w/w), 80 mL) was added to 5ꢀ(4ꢀmethoxyphenyl)ꢀ6ꢀphenylpyridinꢀ2ꢀamine 3a (5.50 g, 19.90
mmol) and the reaction was stirred at 80 °C for 14 h. The mixture was diluted with water (250
mL) and the pH of the solution was adjusted to 8 using solid sodium bicarbonate. The mixture
was extracted with ethyl acetate (2 x 300 mL) and the combined organic extracts were washed
with water (2 x 300 mL) and brine (300 mL). The organic layer was concentrated under reduced
1
pressure to afford 3.71 g (71%) of the product as an offꢀwhite solid. Mp > 250 °C (dec.); H
NMR (CDCl ) δ 4.54 (br s, 2H), 6.54 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 8.1 Hz, 2H), 6.93 (d, J =
3
8
.1 Hz, 2H), 7.20−7.30 (m, 5H), 7.48 (d, J = 8.4 Hz, 1H), 9.36 (br s, 1H); APCIꢀMS (m/z) 263
+
(M+H) .
Intermediates 4b−k were prepared by demethylation of the corresponding 3b−k as described in
the above procedure using hydrobromic acid in glacial acetic acid. Intermediate 4l was prepared
by palladium catalyzed hydrogenolysis of benzyl ether 3l. The characterization data for 4b−l is
given in the supporting information.
6ꢀPhenylꢀ5ꢀ[4ꢀ(quinolinꢀ2ꢀylmethoxy)phenyl]pyridinꢀ2ꢀamine (6a). To a stirred solution of 4ꢀ
(6ꢀaminoꢀ2ꢀphenylpyridinꢀ3ꢀyl)phenol 4a (3.5 g, 13.34 mmol) in DMF (30 mL) were added
cesium carbonate (13.0 g, 39.89 mmol) and 2ꢀ(chloromethyl)quinoline hydrochloride 5a (2.84 g,
13.26 mmol) and the reaction mixture was heated at 70 °C for 48 h. The mixture was diluted
with water (250 mL) and extracted with ethyl acetate (2 x 300 mL). The combined organic
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Journal of Medicinal Chemistry
Page 28 of 61
extracts were washed with water (250 mL) and dried over anhydrous Na SO . The solvent was
2
4
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distilled off under reduced pressure and the residue obtained was purified by flash silica gel
column chromatography using 1% methanol in chloroform to yield 4.19 g (78%) of the product
1
as an offꢀwhite solid. Mp 175–177 °C; H NMR (300 MHz, DMSOꢀd ) δ 5.31 (s, 2H), 6.04 (s,
6
2H), 6.48 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 8.7, 21.0 Hz, 4H), 7.20 (br s, 4H), 7.38 (d, J = 8.4
0
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Hz, 1H), 7.59−7.67 (m, 2H), 7.78 (t, J = 7.2 Hz, 1H), 7.98−8.02 (m, 3H), 8.41 (d, J = 8.1 Hz,
+
1H); HRMS (ESI) calcd for C H N O, [M+H] , 404.1757; found, 404.1758.
2
7
21
3
6ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ[4ꢀ(quinolinꢀ2ꢀylmethoxy)phenyl]pyridinꢀ2ꢀamine (6b). Compound 6b
was prepared by alkylation of phenol 4b with 2ꢀ(chloromethyl)quinoline hydrochloride 5a in the
presence of excess cesium carbonate as described above to give 6b (80%) as an offꢀwhite solid.
1
Mp 186–188 °C; H NMR (DMSOꢀd ) δ 5.32 (s, 2H), 6.06 (s, 2H), 6.49 (d, J = 8.4 Hz, 1H),
6
6.92−7.05 (m, 6H), 7.21−7.26 (m, 2H), 7.38 (d, J = 8.7 Hz, 1H), 7.59−7.67 (m, 2H), 7.78 (t, J =
1
3
8.1 Hz, 1H), 7.98−8.02 (m, 2H), 8.41 (d, J = 8.1 Hz, 1H); C NMR (100 MHz, DMSOꢀd ): δ
6
7
1
1
4
1.2, 107.5, 114.7 (d, J = 21.0 Hz, 2C), 115.0 (2C), 120.0, 123.6, 127.0, 127.6, 128.4, 129.0,
30.3, 130.9 (2C), 132.0 (d, J = 8.0 Hz, 2C), 133.6, 137.4, 137.7, 140.4, 147.4, 153.3, 157.0,
+
58.0, 158.8, 161.7 (d, J = 242.0 Hz, 1C); HRMS (ESI): m/z [M+H] calcd for C H FN O:
2
7
20
3
22.1663; found: 422.1658.
6ꢀ(4ꢀChlorophenyl)ꢀ5ꢀ[4ꢀ(quinolinꢀ2ꢀylmethoxy)phenyl]pyridinꢀ2ꢀamine (6c). Compound 6c
was prepared by alkylation of phenol 4c with 2ꢀ(chloromethyl)quinoline hydrochloride 5a in the
presence of excess cesium carbonate as described above to afford 6c (74%) as an offꢀwhite solid.
1
Mp 188–190 °C; H NMR (300 MHz, DMSOꢀd ) δ 5.33 (s, 2H), 6.10 (s, 2H), 6.50 (d, J = 8.1
6
Hz, 1H), 6.93−7.00 (m, 4H), 7.20−7.28 (m, 4H), 7.39 (d, J = 8.1 Hz, 1H), 7.59−7.63 (m, 2H),
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Journal of Medicinal Chemistry
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.78 (t, J = 8.1 Hz, 1H), 7.99−8.03 (m, 2H), 8.42 (d, J = 8.1 Hz, 1H); HRMS (ESI) calcd for
+
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4
5
6
7
8
9
C H ClN O, [M+H] , 438.1367; found, 438.1364.
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Intermediates 6d−x were prepared by coupling appropriate phenol selected from 4a−l with
appropriate halides or tosylate selected from 5a−g. The characterization data for amines 6d−x is
given in the supporting information.
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N,NꢀDimethylꢀ6ꢀphenylꢀ5ꢀ[4ꢀ(quinolinꢀ2ꢀylmethoxy)phenyl]pyridinꢀ2ꢀamine (7a). To a
stirred solution of 6a (250 mg, 0.620 mmol) in formic acid (5 mL) was added formaldehyde (5
mL) and the reaction mixture was refluxed for 15 h. The mixture was then diluted with water (20
mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were washed
with water (100 mL) and dried over anhydrous Na SO . The solvent was distilled off under
2
4
reduced pressure and the crude material thus obtained was purified by flash silica gel column
chromatography using 30% ethyl acetate in petroleum ether to yield 137 mg (51%) of product as
1
an offꢀwhite solid. Mp 138–140 °C; H NMR (CDCl ) δ 3.15 (s, 6H), 5.35 (s, 2H), 6.54 (d, J =
3
9
2
1
.0 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 7.15−7.21 (m, 3H), 7.35−7.45 (m,
H), 7.47 (d, J = 8.7 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.65−7.77 (m, 2H), 7.83 (d, J = 7.8 Hz,
ꢀ1
H), 8.07 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 9.0 Hz, 1H); IR (neat, cm ): 2924, 1736, 1595, 1496,
+
1428, 1240, 1178, 804; HRMS (ESI): m/z [M+H] calcd for C H N O: 432.2070; found:
2
9
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3
432.2065.
2ꢀ({4ꢀ[6ꢀFluoroꢀ2ꢀ(4ꢀfluorophenyl)pyridinꢀ3ꢀyl]phenoxy}methyl)quinoline (7b). To a stirred
solution of 6b (300 mg, 0.712 mmol) in chloroform (10 mL) were added tetrafluoroboric acid
(0.2 mL, 1.42 mmol) and tertꢀbutyl nitrite (0.3 mL, 2.85 mmol). The reaction mixture was
stirred overnight at room temperature. The mixture was diluted with water (50 mL) and
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