Novel 2-methoxyacylhydrazones as potent, selective PDE10A inhibitors with activity in animal models of schizophrenia

Article abstract of DOI:10.1016/j.bmcl.2012.07.007

A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE10A. Several compounds demonstrated efficacy in animal models of schizophrenia, including conditioned avoidance response and a pro-psychotic phencyclidine hyperactivity model.

Full text of DOI:10.1016/j.bmcl.2012.07.007

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5595–5599
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 2-methoxyacylhydrazones as potent, selective PDE10A inhibitors
with activity in animal models of schizophrenia
Neil S. Cutshall, Rene Onrust, Alex Rohde, Sasha Gragerov, Lauren Hamilton, Kevin Harbol, Hui-Rong Shen,
⇑
Shawn McKee, Charles Zuta, Galina Gragerova, Vince Florio, Thomas N. Wheeler, Jennifer L. Gage
Omeros Corp., 1420 Fifth Ave, Suite 2600, Seattle, WA 98101, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE10A.
Several compounds demonstrated efficacy in animal models of schizophrenia, including conditioned
avoidance response and a pro-psychotic phencyclidine hyperactivity model.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 7 May 2012
Accepted 2 July 2012
Available online 10 July 2012
Keywords:
PDE10
PDE10A
Phosphodiesterase
Schizophrenia
Hydrazone
Hydrazide
PDE
Enzyme inhibitor
Cognitive
Schizophrenia is a debilitating disease affecting approximately
1% of the world population.1 Current pharmaceutical agents used
to treat schizophrenia include the typical antipsychotics that effec-
tively address positive symptoms such as hallucinations and delu-
sions. These drugs exert their effect through action as D2 receptor
antagonists. However, some drawbacks of these medications are
motor side effects and lack of efficacy in a subset of the patient
population.2 Contemporary ‘atypical’ antipsychotics possessing
mixed D2/5HT2A antagonism have reduced severity of motor side
effects, but can cause deleterious metabolic consequences. Besides
the need for new antipsychotic agents without unwanted side ef-
fects, pharmaceuticals are needed that address cognitive symp-
toms which include impairment of working memory and
executive function as well as negative symptoms such as social
withdrawal. The severity of the cognitive effects of schizophrenia
has been shown to be a predominant determinant in the overall
prognosis of patient function. Thus, pharmaceutical entities for
schizophrenia are needed that can manage both positive and neg-
ative symptoms along with cognitive deficits.
predominantly in brain, with highest concentrations in the med-
ium spiny neurons (MSNs) located in the striatum. Because of
the unique location of this enzyme, PDE10A inhibitors have been
studied for their potential as pharmaceutical agents for Parkinson’s
and Huntington’s disease, obsessive compulsive disorder, addiction
and schizophrenia.3b It has been shown that PDE10A inhibitors
have pharmacological effects in models of both the positive and
cognitive symptoms of schizophrenia. The molecular mechanisms
that lead to these in vivo effects continue to be elucidated and
these studies have been reviewed.3b One PDE10A inhibitor, MP-
10, was recently tested for the treatment of schizophrenia in Phase
2a clinical trials where it was found to lack efficacy in acute exac-
erbation of schizophrenia.3b,c It remains to be seen if PDE10 inhib-
itors will be effective in other indications.
We recently reported structure-activity relationship (SAR) stud-
ies on novel thioacetylhydrazones as potent inhibitors of PDE10A
with significant efficacy in animal models of schizophrenia.4 A re-
lated series of compounds also containing the hydrazone function-
ality, but absent the thioether linker is described here. Thus, lead
compound racemic hydrazone 1 was discovered via a high-
throughput screening campaign (Fig. 1). Acylhydrazones have been
pursued as potential therapeutics for cancer (angiogenesis inhibi-
tors),5a inflammatory diseases (cysteine protease inhibitors),5b
type 2 diabetes (glucagon receptor antagonists, taste receptor
modulators),5c,d dementia (calpain inhibitors),5e and HIV (reverse
Phosphodiesterase 10A (PDE10A) is a dual-specificity phospho-
diesterase that hydrolyzes both cyclic adenosine and cyclic guano-
sine monophosphate (cAMP and cGMP).3a It is expressed
⇑
Corresponding author. Tel.: +1 206 676 0813; fax: +1 206 676 0808.
E-mail address: jgage@omeros.com (J.L. Gage).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.007

5596
N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5595–5599
OR2
CN
OH
Region C
O
CHO
O
a or b
c
H
R1
O
R1
R1
N
O
O
N
7
9
8
Region B
Region A
O
O
O
1
H
N
O
PDE10AIC50 359 nM
NH2
d
e
O
O
R1
R1
Figure 1. PDE10A screening hit 1.
10
11
Scheme 2. Reactions and conditions: (a) TMSCN, ZnI2, Et2O, quant., R2 = TMS; (b)
KCN, NaHSO3, EtOAc/H2O, R2 = H; (c) HCl, EtOH, 0° C to rt; (d) NaH, MeI, THF; (e)
NH2NH2, toluene–EtOH (4:3), 105 °C.
transciptase inhibitors).5f Here, we report SAR studies of novel
hydrazone compounds that inhibit PDE10A with low nanomolar
activity, possess excellent PDE selectivity and exhibit oral efficacy
in animal models of schizophrenia.
Table 1
The hydrazones in these studies were synthesized in racemic
form by one of two methods depicted in Schemes 1 and 2. Both
synthetic routes utilized aromatic aldehydes as starting materials
which lent diversity to the number of compounds that could be ac-
cessed. In a typical synthesis, aldehyde 2 was subjected to bromo-
form and potassium hydroxide in MeOH-dioxane6 to give methoxy
phenylacetic acid derivative 3. Esterification of carboxylate 3 fol-
lowed by treatment with hydrazine provided coupling partner acyl
hydrazide 5. This intermediate was reacted with substituted benz-
aldehydes as shown to give 2-methoxyacylhydrazones 6, isolated
solely as trans isomers. These synthetic steps were efficiently con-
ducted with the use of chromatographic purification at only one
step (ester 4). Compounds 12–39, 41–43, and 50–59 were synthe-
sized in this manner.7
A second route to the compounds of interest begins with the
synthesis of a trimethylsilyl cyanohydrin (8, R2 = TMS)8 or cyano-
hydrin (8, R2 = H)9 from benzaldehyde 7 then reaction with hydro-
gen chloride-saturated ethanol9 to give hydroxy phenylacetate 9
(Scheme 2). Alkylation of compound 9 with methyl iodide followed
by reaction with hydrazine in hot toluene-ethanol provided acyl
hydrazide 11. Compounds 40 and 45–46 were synthesized via
the trimethylsilyl cyanohydrin intermediate and compounds 44,
47–49 were synthesized by means of the cyanohydrin.
As part of an initial SAR survey (results not shown) a handful of
compounds were synthesized that led us to discover that the dihy-
drobenzodioxin B region recognition element of analog 12 pro-
vided improved inhibition of PDE10A versus compound 1. Based
on this promising result, SAR studies then embarked upon further
modification of region A while the dihydrobenzodioxin portion of
the molecule was held constant. Table 1 shows some of the com-
pounds synthesized in this series. It has been reported in the liter-
ature that methoxyphenyl rings provide important interactions
with the PDE10A active site.10 It was desired to determine if this
was also the case with the chemical series at hand and if the 3,4-
dimethoxyphenyl moiety of region A could be fine-tuned to in-
crease potency. Therefore, compound 13 was prepared in which
the dimethoxy substituent pattern was rearranged to 3,5-dime-
SAR of region A
R2
O
H
O
O
N
R3
R4
N
O
R5
a
Compd
R2
R3
R4
R5
H
PDE10A IC50 (nM)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
H
H
H
H
H
F
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OEt
OEt
OH
OMe
OMe
OMe
OMe
OMe
OMe
OEt
OMe
H
107
275
28
223
43
12,400
382
3300
150
92
32
19
9
9
128
150
507
92
OMe
OMe
H
OMe
OEt
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Me
H
H
H
H
H
F
Cl
Br
Cl
Br
H
H
H
H
H
H
H
OEt
OMe
OMe
OMe
OMe
OMe
Br
CF3
F
Cl
Br
F
CN
Me
Br
93
2520
6560
14
F
OMe
OMe
OMe
OMe
6
a
Values were calculated from the average of at least two experiments with
mouse PDE10A.11
thoxyphenyl, but this resulted in decreased in vitro activity.
Addition of a third methoxy group (14) increased potency almost
four-fold. Installation of larger alkoxy groups had mixed results.
3,4-Diethoxy analog 15 had reduced activity while 3-ethoxy, 4-
methoxy compound 16 had surprisingly good potency. Removal
of the ether resulted in deterioration of activity (17).
Additional phenyl ring substitutions in region A were also
examined. While fixing the 3,4-dimethoxy substituents on the
phenyl ring, the 2-position was explored. Thus, fluoro-, chloro-
and bromo analogs 18, 19, and 20 were found to exhibit weaker
inhibition. Halogen incorporation at the 5 position was also exam-
ined. Bromo substitution yielded the most potent compound with
this modification (23). Fluoro and chloro substitution (21 and 22)
provided moderate gains in potency. Two compounds which com-
bined the 5-halo motifs with the 3-ethoxy-4-methoxy substitution
pattern were 24 and 25 (IC50 = 9 nM).
O
O
O
O
CHO
O
O
O
O
O
OH
a
b
O
O
4
2
3
O
O
H
N
H
N
O
O
O
O
c
d
NH2
N
R
O
O
6
5
Owing to apprehension that there may be a metabolic instabil-
ity due to demethylation of the 4-methoxy group in region A,4
a
Scheme 1. Reactions and conditions: (a) KOH, CHBr3, MeOH–dioxane; (b) H2SO4,
MeOH, 90 °C, 85% for 2 steps; (c) NH2NH2, EtOH, 90 °C, 95%; (d) ArCHO, HOAc cat.,
EtOH, 80 °C.
replacement was sought. While holding the 3-methoxy substitu-
ent constant, a number of functional groups were placed at the

N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5595–5599
5597
Table 2
SAR of region B
O
H
N
O
Ar
N
O
Br
O
a
a
Compd
Ar
PDE10A IC50 (nM)
Compd
Ar
PDE10A IC50 (nM)
O
34
6.3
45
3.3
N
N
O
O
O
35
36
37
38
10
6.6
7
46
47
48
49
3.4
2.3
2.2
3.9
O
N
N
N
N
N
N
N
12
N
N
N
N
N
39
40
10
50
51
279
10
N
7.6
N
N
N
41
42
3.2
0.7
52
53
4.4
3.6
N
N
O
O
N
O
S
N
N
N
N
N
N
N
N
N
43
10.3
17
54
55
1.7
1.3
O
N
S
O
44
N
N
a
Values were calculated from the average of at least two experiments with mouse PDE10A.
4-position (see 26–30). The most beneficial substitution proved to
be with chloro and bromo groups as exemplified by compounds
29 and 30 with IC50 values below 100 nM. Replacement of both
alkoxy substituents was unproductive (31 and 32). Finally, two
compounds were synthesized with variations at the 4-position
and methoxy substituents at each of the 3- and 5-positions.
Hydrazones 33 and 34 demonstrated good enzymatic activity at
14 and 6 nM, respectively.
Subsequently, region A of optimized lead 34 was fixed while the
impact of changes in region B was explored (Table 2). Monocyclic,
bicyclic and fused bicyclic ring systems in this series were investi-
gated. It was found that benzodioxole 35 had nearly equivalent
activity to 34. Dimethylamine, pyrrolidine and N-methylpiperazine
susbstitution at either the 3- or 4-phenyl positions was well toler-
ated in most cases, but did not improve activity (36–40). Morpho-
line installation gave a two-fold improvement in potency (see 41).
Cyclic sulfonamide 42 was the most active compound with an IC50
of 0.7 nM. Sulfone derivative 43 also did not improve activity.
The impact on inhibition by replacement of the phenyl ring in
region B with an aromatic heterocycle was next examined. An
unsubstituted pyridine was not beneficial while morpholinyl- or
piperidinyl-substituted pyridine gave improved potency (45–46).
Fusion of the meta-pyridyl moiety to give a quinoline (47) also im-
proved activity as compared to lead compound 34. Other quinoline
examples had similar potency (48–49) while benzimidazole 50
was less active. Lastly, bicyclic aromatic systems were studied.

5598
N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5595–5599
Table 3
SAR of region C
140
120
R2
O
H
N
O
N
100
O
R1
Br
80
O
60
a
PDE10A IC50 (nM)
40
Compd
R1
R2
41
56
57
55
58
59
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
1H-Pyrazol-1-yl
1H-Pyrazol-1-yl
1H-Pyrazol-1-yl
Me
Et
3.2
1.9
1.7
20
iPr
Me
Et
1.3
2.5
0
iPr
8.3
INT 1
INT 2
INT 3
INT 4
INT 5
Intervals 4 min each
a
Values were calculated from the average of at least two experiments.
vehicle + PCP
55-5 + PCP
61-4 + PCP
Table 4
SAR and PK of separated enantiomers
Figure 3. Compounds 55 and 61 reduced PCP-induced hyperactivity in mice when
administered orally at 5 or 4 mg/kg as indicated.15
O
H
N
O
N
2-isopropoxy (57) substitution offered a small improvement in po-
tency. However, in comparison to compound 55, the 2-ethoxy (58)
and 2-isopropoxy (59) analogs were slightly less potent.
O
Br
R
O
Preparatory chiral HPLC was conducted on racemic compounds
41 and 55 to resolve the enantiomers.12 The first-eluting enantiomer
was the most active in both cases (compounds 60 and 61). All of the
hydrazone-based compounds in these studies were found to possess
adequatein vitro stability in simulated gastric fluid (data not shown)
therefore they were tested in pharmacokinetic (PK) studies. The
PDE10A inhibitory activities, as well as several PK parameters mea-
sured are reported in Table 4. The oral bioavailability (F) in mice was
good to excellent while the brain/plasma ratios were low.
Compd
R
Chirality
PDE10A IC50, nM
%F
B/P (m)
41
60
55
61
4-Morpholinyl
4-Morpholinyl
1H-Pyrazol-1-yl
1H-Pyrazol-1-yl
Racemate
Enant. 1a
Racemate
Enant. 1a
3.2
0.64
1.3
0.8
63
91
48
92
0.37
0.27
0.39
0.12
Enant 1 refers to the first eluting enantiomer on a chiral HPLC column.12
a
The selectivity ofseveral compounds inthis seriesfor PDE10Aver-
sus other PDEs was investigated. It was found that several key com-
pounds (41, 55, 60–61) possessed >300-fold selectivity over PDE
isoforms PDE1A-PDE11A (excluding PDE6A which was not tested).
A subset of the most potent compounds in this series was tested
for in vivo activity in mouse behavioral models. The conditioned
avoidance response (CAR) assay is widely believed to be predictive
of antipsychotic activity.13 Drugs possessing this activity reduce
number of avoidances in this test. Racemates 41 and 55 demon-
strated modest activity in this assay when administrated orally
at 10 mg/kg in mice (Fig. 2). The active enantiomers of these com-
pounds, 60 and 61, had equivalent or improved in vivo potency
when administered at half the dosage. It is not understood why
compound 61 had better activity than 60 given that the in vitro
activity and bioavailability were very similar. Pro-psychotic phen-
cyclidine (PCP) causes hyperactivity in rodents that can be reduced
by administration of known antipsychotic drugs. Thus, this model
is considered to be predictive of antipsychotic activity.13 Both race-
mate 55 and enantiomer 61 reduced hyperlocomotion at 5 or
4 mg/kg (oral administration) in this model (Fig. 3).
120
100
80
60
40
20
0
vehicle 41-10
60-5
55-10
61-5
Figure 2. Compounds 41, 60, 55 and 61 attenuated conditioned avoidance response
in mice when administered orally at 5 or 10 mg/kg as indicated.14
Compound 61 was also tested in a novel object recognition as-
say (data not shown). PCP disrupts animal performance in this
memory test.13 Unlike olanzapine, which was unable to restore
discrimination between a familiar and a novel object, compound
61 was successful in apparent memory restoration when adminis-
tered 4 mg/kg in mice IP.16 This experiment points to a possibility
that unlike current antipsychotics, PDE10A inhibitors may be able
to address cognitive symptoms of schizophrenia.
These changes proved to be productive in most cases (52–55). Spe-
cifically, N-pyrazole analog 55 was one of the most active
compounds.
In parallel to the SAR efforts which focused on the distal por-
tions of the molecules, variations in the linker region C were also
examined. Table 3 describes the PDE10A activity of several active
acyl hydrazones in which the 2-methoxy substituent was modi-
fied. In comparison to compound 41, both the 2-ethoxy (56) and
In conclusion, SAR studies of regions A, B and C of original lead
compound 1 followed by chiral separation provided enantiomers

N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5595–5599
9. Zymalkowski, V. F.; Tinapp, P. Justus Liebigs Ann. Chem. 1966, 98.
5599
60 and 61, both with subnanomolar inhibition of PDE10A in vitro.
Analogs 60 and 61 have excellent oral bioavailability and are active
in the CAR mouse model despite low brain/plasma ratios. Com-
pound 61 was also active in a hyperactivity assay and a memory
model.
10. (a) Helal, C. J.; Kang, Z.; Hou, X.; Pandit, J.; Chappie, T. A.; Humphrey, J. M.;
Marr, E. S.; Fennell, K. F., et al J. Med. Chem. 2011, 54, 4536.; (b) 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. J.
Med. Chem. 2007, 50, 182.
11. PDE10A assay. Compounds were tested for PDE10A potency by measuring the
inhibition of PDE10A hydrolysis of [3H]cGMP to [3H]GMP. Generally, eight
dilutions of compound were assayed in 50 mM Tris–HCl pH 7.5, 8.3 mM MgCl2,
0.5 mg/mL BSA, 1.7 mM EGTA, 16 nM [3H]cGMP and 1% DMSO. To start the
Acknowledgement
reaction, mouse PDE10A (BPS BioSciences, CA) was added to
a final
The authors would like to thank the Stanley Medical Research
Institute for generous financial support of this program.
concentration of 25 ng/ml. The reaction was incubated at 30 °C for 20 min.
PDE10A hydrolysis was terminated by the addition of yttrium silicate beads
(GE Healthcare, RPNQ0150) and counted on a Wallac Microbeta scintillation
counter, 1–2 h following the addition of the beads. Data was analyzed using
XLfit (Microsoft) from which IC50 values were obtained.
References and notes
12. Chiral preparatory HPLC was performed on a Waters Alliance 2695 instrument
equipped with a Waters 2996 diode array detector and a Regis Tech., Inc. Chiral
(R,R) WHELK-01 10/100, 10.0 Â 150 mm column at 25 °C; mobile phase 100%
ethanol. Detection wavelength was 303 nM. Run time was 15 min for
compound 41; peak 1 (7.1 min), peak 2 (13.0 min). Run time was 11 min for
compound 55; peak 1 (5.3 min), peak 2 (8.2 min).
13. Castagné, V.; Moser, P. C.; Porsolt, R. D. Adv. Pharmacol. 2009, 57, 381.
14. Mice were trained to avoid foot-shock by moving to the other side of a shuttle-
box in response to conditional stimulus (CS). Training proceeded until mice
demonstrated at least 25 avoidances out of 30 trials. Compounds were
administered by oral gavage in 10% Vitamin E-TPGS + 5% DMSO 20 min before
testing. Control group received vehicle injection. Eight mice per group were
used. Paired-sample t-test was used to compare performance of the same
animals on the day of the experiment and on the previous day when vehicle
was administered. p <0.05 For all doses and compounds. Data shown are
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Majchrzak, M. J.; Ramirez, A. D.; Schmidt, K.; Seymour, P. A.; Siuciak, J. A.;
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combined from multiple experiments and represent avoidances as
percentage of the vehicle control.
a
15. Activity was monitored by number of beam breaks in an open field novel
environment. Compound (4 or 5 mg/kg) or vehicle (30 mM H3PO4, 0.2% tween
80, 5% DMSO) were administered orally 20 min before testing, and PCP was
injected by the same route 10 min before testing. Eight mice per group were
used, t-test for each interval showed p <0.005. Data shown are combined from
multiple experiments and represent the percent of activity of vehicle control.
16. Singly housed mice were coinjected (IP) with vehicle or compound 61 (4 mg/
kg) and PCP (5 mg/kg). Twenty minutes after dosing, 2 identical objects were
placed in the cage for 40 min then removed. Twenty-two hours later, one of the
objects from day 1 and one novel object were placed in the cage. Once the
animal approached the objects, time spent with each object was recorded for
4 min. Results are reported as % time spent with novel object [time with novel
object (seconds)/total time (seconds) X100].
6. Brittain, D. R.; Longridge, J. L.; Preston, J.; Salter, L.; Morris; J. J.; Cooper, A. L.;
Brown, S. P. U.S. Patent 5270342 A1, 1993.
7. Methyl ester 66, precursor to cyclic sulfonamide 42, was synthesized as shown
below.
8. Schnur, R. C. U.S. Patent 4,332,952, 1982.
O
O
H2
10% Pd/C
O
Scheme1
H
O
O
O
O
EtOH
O2N
O2N
H2N
62
63
64
O
O
O
O
Cl
O
Cl
S
O
O
N
O
O
O
O
S
N
Cl
O
S
O
pyridine
DMF
50 °C
N
H
65
66
O
H
O
O
TMSN
3
O
1) THF
HCl
O
O
64
O
N
2) PPh , Et N
3
3
MeOH
O
N
N
CN
CCl
4
N
CH Cl
2
2
67
68
Methyl ester 68, precursor to tetrazole 53, was synthesized from common intermediate 64 as shown.