Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties

Article abstract of DOI:10.1021/acs.jmedchem.7b00302

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

Full text of DOI:10.1021/acs.jmedchem.7b00302

Article
pubs.acs.org/jmc
Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory
Enhancing Properties
Brian Dyck,*,† Bryan Branstetter,† Tawfik Gharbaoui,† Andrew R. Hudson,† J. Guy Breitenbucher,†
Laurent Gomez,† Iriny Botrous,† Tami Marrone,† Richard Barido,† Charles K. Allerston,†
E. Peder Cedervall,† Rui Xu,† Vandana Sridhar,† Ryan Barker,† Kathleen Aertgeerts,† Kara Schmelzer,†
David Neul,† Dong Lee,† Mark Eben Massari,† Carsten B. Andersen,† Kristen Sebring,† Xianbo Zhou,†
Robert Petroski,† James Limberis,† Martin Augustin,§ Lawrence E. Chun,‡ Thomas E. Edwards,‡
Marco Peters,† and Ali Tabatabaei†
†Dart Neuroscience LLC, 12278 Scripps Summit Drive, San Diego, California 92131, United States
‡Berylllium, 7869 NE Day Road West, Bainbridge Island, Washington 98110, United States
§Proteros Biostructures GmbH, Bunsenstraße 7a, D-82152 Martinsried, Germany
S
* Supporting Information
ABSTRACT: A series of potent thienotriazolopyrimidinone-
based PDE1 inhibitors was discovered. X-ray crystal structures
of example compounds from this series in complex with the
catalytic domain of PDE1B and PDE10A were determined,
allowing optimization of PDE1B potency and PDE selectivity.
Reduction of hERG affinity led to greater than a 3000-fold
selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-
((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydro-
pyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-
5(6H)-one was identified as an orally bioavailable and brain
penetrating PDE1B enzyme inhibitor with potent memory-
enhancing effects in a rat model of object recognition memory.
therapeutic intervention in a variety of peripheral disorders.
Among them, PDE5 inhibitors marketed for the treatment of
erectile dysfunction and pulmonary artery hypertension have
shown the most success (sildenafil, tadalafil, vardenafil). In
addition, PDE4 inhibitors have been approved for the
treatment of COPD and psoriasis (roflumilast, apremilast)
and PDE3 inhibitors for acute treatment of congestive heart
failure (milrinone).
Because of the putative impact of cAMP and cGMP on
neuronal plasticity, PDE inhibitor have potential utility for CNS
disorders.6,7 The second messenger cAMP activates cAMP
response element-binding protein (CREB) signaling via protein
kinase A (PKA) and can thereby modulate transcription of
genes associated with synaptic plasticity, such as brain-derived
neurotrophic factor (BDNF).8 This cAMP/PKA/CREB path-
way has been implicated in the enhancement of long-term
potentiation (LTP), which is considered the neurophysiological
correlate of memory.9−11
INTRODUCTION
■
Cognitive decline and loss of memory with age has a significant
impact on daily functioning and quality of life.1 These deficits
are even more prominent in many neurodegenerative and
psychiatric disorders such as depression, Alzheimer’s disease,
Huntington’s disease, and schizophrenia. Plainly, there is a
significant need for new drugs which ameliorate these cognitive
deficits. Most drug discovery efforts to intervene in cognitive
dysfunction have focused on neurotransmitter systems thought
to be involved in cognitive functioning, including acetylcholine,
serotonin, histamine, glutamate, and dopamine systems.2
However, an alternative approach is to target enhancement of
the downstream signaling cascades that result from activation of
these neurotransmitter systems, which trigger activity-depend-
ent gene expression and support long-lasting changes in
synaptic structure and function.3
cAMP and cGMP are ubiquitous second messengers which
are upregulated upon neuronal activation. Phosphodiesterases
(PDEs) are the enzymes which hydrolyze, and thereby
inactivate, these second messengers, making the PDEs essential
for the termination of cGMP and cAMP signaling. Eleven
families consisting of 21 mammalian genes encoding individual
PDE isoforms have been identified.4,5 Because of their critical
role in signal transduction, PDEs are key molecular targets for
Among the PDEs expressed in the CNS, the dual specificity
PDE1B may play a unique role pertaining to memory.12,13 Like
PDE4 and PDE10, inhibition of PDE1B increases cAMP levels
Received: February 22, 2017
© XXXX American Chemical Society
A
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in the striatum.12,14 PDE1 is coexpressed with dopamine
receptors, as are PDE4 and PDE10, suggesting these PDEs are
responsible for terminating the cAMP signal associated with
dopaminergic activation. However, Nishi et al. have proposed
PDE4 and PDE10 activate DA neurons predominantly via the
indirect D2 pathway and PDE1B couples with the direct
pathway via D1 signaling.15 This offers an explanation why a
PDE1B inhibitor reversed haloperidol-induced catalepsy in rats,
whereas inhibitors of PDE4 and PDE10 enhance catalepsy
induced by haloperidol.16a Consequently, a PDE1B inhibitor
may be well suited to treat cognitive and motor deficits
associated with deficient D1 dopaminergic signaling such as
exists in Parkinson’s disease.
efficacy in a rat novel object recognition (NOR) task at 1 and 3
mg/kg (PO), and has advanced into the clinic in partnership
with Takeda.24 Recently, scientists from Pfizer have also
disclosed a brain-penetrating quinazoline-based PDE1 inhibitor
PF-04822163 (PDE1B IC50 0.0024 μM), but no efficacy data
was reported.25
RESULTS AND DISCUSSION
■
Screening our compound collection for PDE1B inhibitory
activity generated the triazolopyrimidinone lead 3. Despite
cross-reactivity with several PDEs, including cGMP-specific
PDE5 and PDE6, the low molecular weight and polar surface
area of 3 suggested it may make a suitable scaffold for
optimization (Table 2). Attempts to model 3 within the
published apo PDE1B structure (accession code 1TAZ) were
unsuccessful, so a crystal structure of this compound bound to
the catalytic domain of PDE1B was determined (Figure 1).26
The reason for the difficulty encountered while trying to
model 3 within the ligand-free PDE1B structure became clear
upon obtaining a crystal structure of the complex. The carbonyl
of compound 3 forms hydrogen bonds with the side chain of
Gln421 and His373, and there is a favorable interaction
between the triazole nitrogen N(1) and the side chain of
His373. This orientation of the ligand places the 4-chlorobenzyl
group into a newly formed lipophilic pocket which arises from a
rotation of Met389 relative to the apo structure of the protein.
Phe424 and Leu388 form the hydrophobic clamp typical of
PDE structures. The cyclohexyl ring of 3 protrudes into a
solvent exposed region, which we thought to exploit to
optimize physicochemical properties of the compound and
perhaps impart additional selectivity over other PDEs. One
option we felt was attractive was to use a fused piperidine ring
in replacement of the cyclohexyl group due to its higher water
solubility and chemical versatility.
Few selective PDE1 inhibitors have been disclosed thus far
(Chart 1). Vinpocetine, a commonly used nutraceutical with
Chart 1. PDE1B Inhibitors
putative ameliorating effects on primary dementia, has minimal
activity at PDE1B in our hands.17 The modest PDE1 potency
and off-target activity of this compound suggest that experi-
ments using vinpocetine to determine PDE1 pharmacology
should be interpreted with caution. Other potent PDE1
inhibitors have been reported with cross-reactivity with
PDE5, PDE9, and PDE10.18−22 Scientists from Schering-
Plough have optimized the dual PDE1/PDE5 inhibitor SCH-
51866 (PDE1 IC50 0.070 μM, PDE5 IC50 0.060 μM) to
compounds with greater than 300-fold selectivity for PDE1
over PDE5 (e.g., 2).19,23 Intra-Cellular Therapies has identified
potent PDE1 inhibiting pyrazolopyrimidinones structurally
related to the Schering-Plough imidazopyrimidinones.16 One
example (ITI-214, PDE1B IC50 0.000058 μM) demonstrated
The synthetic route used to prepare the piperidinothiophene
analogues of 3 is shown in Scheme 1. Reaction of protected
piperidinones 4 and 5 with elemental sulfur and malonitrile
under the conditions described by Wang and co-workers
afforded 6 and 7.27 These were converted to the ethyl
carbamates and heated with formylhydrazine to afford
tectracyclics 8 and 9.28 Substituted benzyl groups were
introduced by N-alkylation and the t-butoxycarbonyl group
was removed by the action of trifluoroacetic acid, yielding
piperidines 10−15. Substitution on the piperidine nitrogen
atom was accomplished by reductive alkylation with an
Figure 1. Crystal structure of 3 bound to the catalytic domain of PDE1B and ligand interaction guide. Key residues are labeled, including the
hydrophobic clamp (Phe424 and Leu388).
B
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(and lipophilic) channel formed by Phe729 and Met713. The
pKa of 16j was determined experimentally to be 6.6, so a
significant fraction of the aminergic inhibitors would be
expected to be protonated at physiological pH. It is possible
the narrow, lipophilic channel does not tolerate ionized groups.
In any case, a basic nitrogen atom at this site proved to be a
good approach to achieve selectivity over PDE10.
Scheme 1. Synthesis of Thienopyrimidinone PDE1
Inhibitors
a
In terms of PDE1B inhibition, alkyl (16b), benzyl (16f), and
cyclic ether-substitution (16e and 16h) all provided potent
examples, but minimizing hERG activity while retaining a basic
nitrogen within the piperidine ring proved challenging. A 4-
substituted benzyl group on the pyrimidinone nitrogen atom
improved activity at PDE1B as 16g was less potent, and
fortunately modifications did allow attenuation of hERG
activity. Replacement of the 4-chloro group with a 4-methoxy
as in 16j led to a 6-fold reduction in hERG potency. An
additional ortho substituent on the benzyl ring as in 16i and
16k had a modest positive effect on hERG activity as well, but
the ratio of hERG IC50 to PDE1B IC50 remained largely
unchanged at 80-fold. A two log reduction of the pKa of the
piperidine nitrogen atom by β-difluorination as in 17 resulted
in a 9-fold improvement in PDE1B potency and a 4-fold
increase in the hERG IC50. Consequently, 17 exhibited greater
than a 3000-fold ratio between its PDE1B IC50 and hERG IC50.
Because of the modest hERG inhibition observed for these
compounds, representatives were tested in anesthetized guinea
pigs for signs of QT prolongation. No QT prolongation was
observed for 16k or 17 up to doses of 10 mg/kg IV (Figure 3).
This suggests that the 80-fold ratio between in vitro hERG and
PDE1 activities is probably sufficient to provide a safe margin in
vivo (see efficacy doses below). Unfortunately, difluoropiper-
idine 17 was a substrate of MDR1 (P-gp) in MDCK cells with
an efflux ratio greater than five, thereby precluding it from
advancing as a candidate for CNS indications.
A crystal structure of 16j with the catalytic domain of PDE1B
was determined and the binding mode is similar to that of the
screening hit 3 (Figure 4). The tetrahydropyranylmethyl group
of 16j protrudes into a pocket comprised of Leu409, Pro408,
Phe392, and Thr271. It should be noted that the position of the
tetrahydropyranylmethyl group could potentially be influenced
by a second molecule of 16j that is bound between two crystal
symmetry related protein molecules, next to the active site of
the PDE1B molecule in the asymmetric unit (see Supporting
Information, Figure S1)
By improving PDE1B potency, the tetrahydropyranylmethyl
group of 16k imparted good selectivity with regard to other
PDE families (Table 2). This is especially evident for the PDE6
isoform, in which the IC50 ratio for 16k shows more than a 200-
fold improvement relative to 3. PDE6 expression is largely
limited to the human retina and inhibition of this enzyme may
be responsible for some of the visual side effects of marketed
erectile dysfunction drugs.29
a
Reagents and conditions: (a) malonitrile, sulfur, proline, DMF, 60
°C; (b) ethyl chloroformate, pyridine, DCM, then formyl hydrazine,
DIEA, DMA, 150 °C; (c) ArCH2Cl, K2CO3, DMF, 60 °C; (d) TFA,
DCM; (e) aldehyde/ketone, NaBH(OAc)3, DCE or cyclopropylcar-
bonyl chloride, TEA, DCM or 2,2-difluorocyclopropanecarboxylic
acid, TEA, HOBt, EDC, DCM.
aldehyde or ketone or acylation under standard conditions,
thereby affording target compounds 16a−k and 17.
The activity of the piperidines 16a−k and 17 at PDE1B is
shown in Table 1, along with in vitro microsomal stability data
and inhibitory data against two antitargets, PDE10 and hERG.
As predicted by the crystal structure shown in Figure 1,
substitution of nitrogen for carbon in the cyclohexyl ring did
not negatively influence PDE1B activity, as 3 and 16a exhibit
similar potency at PDE1B. We did however observe a
substantial increase of potency at the hERG channel, which
may not have been unexpected with the addition of a tertiary
amine functionality. Substitution of the nitrogen atom with
cyclopropylmethyl (16b) resulted in a 5-fold increase in
potency at PDE1B and a modest improvement in hERG
activity. Rendering the molecule nonbasic by introducing an
amide functionality into 16c increased the potency 4-fold at
PDE1B relative to 16b and reduced the hERG potency by 3-
fold. Unfortunately, acyl substituted piperidines consistently
inhibited PDE10A. In fact, this PDE10 activity was optimized
to produce potent dual PDE1/PDE10 inhibitors such as 16d.
In an effort to understand the origin of the cross-reactivity with
PDE10, we determined a crystal structure of the complex of
16d with the catalytic domain of PDE10A (Figure 2).
For the most part, the thienotriazolopyrimidinones were
stable in human and rat liver microsomes (Table 1). The
exceptions were the methyl and cyclopropyl substituted
piperidines 16a and 16b which were rapidly cleared in the
microsomes from both species and benzyl substituted 16f,
which had moderate stability in human microsomes, but not rat
microsomes. Fortunately, 16k had good stability in the in vitro
assay for both species and is not a P-gp substrate (MDCK efflux
ration = 1.0). Hence, the compound was evaluated in rats orally
and intravenously (Figure 5).
The binding mode of 16d in PDE10A is inverted relative to
that of similar compounds bound to PDE1B. A triazole
nitrogen atom forms a hydrogen bond with the conserved
glutamine (Gln726). The hydrophobic clamp of PDE10A
(Phe729 and Ile692) encloses the tetracyclic core, and the
carboxamide side chain extends distally from the metal centers.
From the PDE10 crystal structure, it is difficult to ascertain why
the compounds containing a carboxamide functionality are
consistently more potent at PDE10 than the corresponding
amines. This portion of the inhibitor does occupy a narrow
C
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Table 1. In Vitro Properties of Key Compounds
a
b
Values are averaged from a minimum of three replicates. Standard error of the mean (SEM) values were typically less than 40%. Intrinsic in vitro
c
clearance determined in human liver microsomes (HLM) in the presence of NADPH. Intrinsic in vitro clearance determined in rat liver microsomes
(RLM) in the presence of NADPH.
Figure 2. Crystal structure of 16d bound to PDE10A and ligand interaction guide. Key residues are labeled, including the hydrophobic clamp
(Phe729 and Ile692). HID, δ histidine.
D
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Table 2. PDE Selectivity Data for 3 and 16k
compd 3
compd 16k
a
,
b
a,b
IC50
PDE1B
selectivity
IC50
PDE1B
selectivity
(μM)
(μM)
PDE1B
0.46
4.1
1
9
0.026
7.9
1
>200
>200
>200
10
PDE2A1
PDE3A
>10
3.9
>20
8
>10
PDE4D3
PDE5A
9.3
0.37
0.46
>10
>10
>10
4.0
1
0.26
PDE6C
1
>10
>10
8.4
>10
0.74
0.28
>200
>200
>200
>200
29
PDE7A1
PDE8A1
PDE9A2
PDE10A1
PDE11A4
>20
>20
>20
9
2.3
5
11
a
PDE1B, PDE2A1, PDE3A, PDE4D3, and PDE10A1 IC50 values are
averaged from a minimum of three replicates. Standard error of the
b
mean (SEM) is typically less than 40%. PDE5A, PDE6C, PDE7A1,
PDE8A1, PDE9A2, and PDE11A4 IC50 values were determined at BPS
Bioscience.
Figure 3. Effect of PDE1 inhibitors 16k and 17 on the QT interval of
anesthetized guinea pigs. (A) 16k infused at 0, 1, 3, and 10 mg/kg
(IV). (B) 17 infused at 0, 1, 3, and 10 mg/kg (IV).
Pharmacokinetic properties of 16k were evaluated in male
SD rats (200−250 g) following administration of a single 1 mg/
kg IV or 5 mg/kg PO dose formulated as a solution in a mixture
of NMP:PEG400:water (10:40:50). The compound was
characterized as having a high volume of distribution (Vss)
and a moderate systemic clearance (50% of liver blood flow).
The elimination half-life was determined to be 2.3 h. Following
oral administration, 16k was rapidly absorbed with a Tmax of 0.8
h. It was found to be orally bioavailable (F = 90%) in rats and
penetrate the brain (B/Pc,max = 1.7). Compound 16k was 1.6%
unbound in rat brain tissue and 7.2% unbound in rat plasma.
On the basis of this data, the compound was evaluated in a test
of rat novel object recognition (NOR, Figure 6), a benchmark
test of recognition memory in rodents.30 In this paradigm, rats
were allowed to explore two identical objects in the training
phase. In the testing period, 24 h later, the rats were presented
with one of the objects encountered during training (the
Figure 5. Rat plasma concentrations of 16k following either a single 5
mg/kg PO or 1 mg/kg IV administration in 10:40:50 NMP:PEG400:-
water.
familiar object) and a novel object. The natural curiosity of the
animal caused it to spend more time exploring the new object.
Measurement of the time spent with this novel object at the
expense of the familiar object allowed one to quantify memory
of the latter. A drug that improves memory should ideally
augment performance of rats given suboptimal training to a
Figure 4. Crystal structure of 16j bound to the catalytic domain of PDE1B and ligand interaction guide. Key residues are labeled, including the
hydrophobic clamp (Phe424 and Leu388). The carbonyl group and N(1) of compound 3 form a hydrogen bond network with His373 and Gln421.
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Figure 6. Effect of 16k in a rat model of recognition memory. (A) Experimental procedure. Rats were habituated for 3 days. On day four, vehicle or
drug was given and the rats trained for 3 min to induce a weak memory against which a drug effect could be measured. A separate group of rats was
treated with vehicle and trained for 15 min as a performance control. (B) Effect on 24 h memory retention. Rats treated with 0.3 mg/kg 16k showed
significantly stronger memory for the familiar object than rats treated with vehicle and trained for 3 min. Memory in this group was comparable to
the 15 min training + vehicle control. *p < 0.05 vs vehicle. (C) Effect of drug on exploration of the novel and familiar object during memory testing.
Rats treated with 0.3 mg/kg 16k, but not vehicle treated rats, spent significantly more time exploring the novel object. *p < 0.05 for novel vs familiar.
(D) 16k did not affect object exploration during training. (E) 16k did not affect locomotion during training. All groups N = 18−22. The mean
SEM are shown.
comparable level as untreated animals trained to induce a
strong memory (i.e., more exposure to objects during the
training phase). Studies in monkeys and rodents indicate that
the hippocampus and cortical structures are important for
recognition memory, and both areas show high levels of PDE1
expression.31−36 We dosed rats orally with 16k or vehicle 60
min prior to training and trained for 3 min to induce a weak
memory for the trained object (Figure 6A). Rats treated with
0.3 mg/kg 16k showed significantly enhanced recognition
memory compared to vehicle treated rats in a retention test
given 24 h later (p < 0.05, Figure 6B). At this dose, the
maximum brain and free brain concentrations are estimated to
be 110 and 1.8 nM, respectively. Memory in 0.3 mg/kg 16k
treated rats was comparable in magnitude to that observed in
rats treated with vehicle and trained for 15 min (strong
training), and both groups spent significantly more time
exploring the novel object on test whereas vehicle treated rats
trained for 3 min did not (Figure 6C). Importantly, 16k did not
affect the time exploring the objects or locomotion in the
training phase, indicating that the drug did not alter exploratory
behavior in rats (Figure 6D/E).
CONCLUSION
■
cAMP signaling is fundamental to memory formation. A
number of access points to this important second messenger
may be susceptible to pharmacological intervention, and
phosphodiesterase inhibition is among the most studied. We
have identified a series of selective PDE1 inhibitors with good
pharmacokinetic properties and brain penetration to test
memory effects in rats. Among these inhibitors, 16k (DNS-
F
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(t, J = 5.65 Hz, 2H), 2.93−3.02 (m, 2H), 1.45 (s, 9H). LCMS (MH+,
348.2).
0056) increases long-term memory in a rat model of
recognition memory, at brain concentrations near the PDE1B
IC50.37 This compound shows potential as a tool to further
probe the pharmacology of PDE1 inhibition. Moreover, related
PDE1 inhibitors have promise as therapeutics to treat memory
deficits and other conditions secondary to aberrant dopami-
nergic signaling.
tert-Butyl 11,11-Difluoro-5-oxo-5,6,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-9(8H)-car-
boxylate (9). The title compound was prepared using general method
1
A. H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 7.39 (d, J = 8.53
Hz, 2H), 6.94 (d, J = 8.53 Hz, 2H), 5.37 (s, 2H), 4.74 (br s, 2H),
4.05−4.18 (m, 2H), 3.75 (s, 3H), 1.43 (s, 9H). LCMS (MH+, 504.2).
6-(4-Chlorobenzyl)-5-oxo-5,6,8,9,10,11-hexahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-9-ium 2,2,2-
trifluoroacetate (10). General method B: Potassium carbonate (1.59
g, 11.5 mmol) was added to a mixture of 8 (2.00 g, 5.76 mmol) and 4-
chlorobenzyl chloride (1.11 g, 6.91 mmol) in DMF (20 mL). The
resulting mixture was heated to 55 °C for 20 h, cooled to room
temperature, and poured into water (200 mL) with vigorous stirring.
The resulting precipitate was isolated by filtration and air-dried to
afford 2.40 g (88%) of 6-(4-chlorobenzyl)-5-oxo-5,6,10,11-
tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]-
EXPERIMENTAL SECTION
■
General Methods. Solvents and reagents were purchased and used
without further purification unless otherwise noted. Vinpocetine (1)
was purchased from Sigma-Aldrich. Compounds 2 and 6 were
prepared as previously described.19,27 Flash column chromatography
(FCC) was performed on Teledyne Isco CombiFlash instruments
equipped with silica gel columns. Preparative HPLC purification was
performed on a Shimadzu Prominence system equipped with a Waters
Sunfire OBD C18 5 μM 30 mm × 100 mm column. Elution was
achieved with increasing concentration of acetonitrile in water and
0.05% trifluoroacetic acid added as modifier. Analytical HPLC was
performed on an Acquity UPLC system equipped with a BEH C18 1.7
μM 2.1 mm × 50 mm column. Elution was achieved with increasing
concentration of acetonitrile in water and 0.1% formic acid added as
modifier. NMR was performed on a Bruker Ultrashield 400 Plus
spectrometer. All final compounds gave satisfactory results ( 0.4%)
upon combustion analysis at Robertson Microlit Laboratories. QT
prolongation studies in anaesthetized guinea pigs was performed by
CorDynamics.
1
pyrimidine-9(8H)-carboxylate as an off-white powder. H NMR (400
MHz, CDCl3) δ 8.32 (s, 1 H), 7.41−7.45 (m, 1H), 7.34−7.37 (m,
2H), 5.39 (s, 2H), 5.39 (s, 2H), 4.59−4.69 (m, 2H), 3.74−3.82 (m,
2H), 3.11−3.21 (m, 2H), 1.51 (s, 9H). LCMS (MH+, 472.1).
The above material (1.70 g, 3.6 mmol) was dissolved in DCM (20
mL) and treated with TFA (5 mL). The resulting mixture was stirred
at room temperature for 3 h, and it was concentrated under reduced
1
pressure to yield 10 (1.68 g, 96%) as the trifluoroacetic acid salt. H
NMR (400 MHz, DMSO-d6) δ 9.28 (br s, 2H), 8.55 (s, 1H), 7.44 (d, J
= 2.76 Hz, 4H), 5.42 (s, 2H), 4.36 (br s, 2H), 3.43−3.55 (m, 2H),
3.16−3.26 (m, 2H). LCMS (MH+, 372.1).
6-(Benzyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one Trifluoroacetate (11). The
title compound was prepared using general method B. 1H NMR (400
MHz, DMSO-d6) δ 9.28 (br s, 2H), 8.55 (s, 1H), 7.44−7.30 (m, 5H),
5.42 (s, 2H), 4.35 (s, 2H), 3.49 (t, J = 5.9 Hz, 2H), 3.21 (t, J = 5.6 Hz,
2H). LCMS (MH+, 338.1).
tert-Butyl 2-Amino-3-cyano-4,4-difluoro-4,5-dihydrothieno[2,3-
c]pyridine-6(7H)-carboxylate (7). tert-Butyl 3,3-difluoro-4-oxopiper-
idine-1-carboxylate (5.10 g, 20 mmol), malononitrile (1.46 g, 22
mmol), sulfur (0.97 g, 30 mmol), and DL-proline (0.23 g, 2.0 mmol)
were combined in DMF (25 mL) and heated at 65 °C for 18 h. The
mixture was concentrated under vacuum and purified by FCC (elution
with 10−80% ethyl acetate in hexanes) to afford 2.22 g (35%) of 7 as a
yellow oil which solidified on standing. 1H NMR (400 MHz, CDCl3) δ
4.47 (br s, 2H), 4.13 (q, J = 7.11 Hz, 1H), 3.98 (t, J = 10.54 Hz, 2H),
1.45−1.53 (m, 9H), 1.26 (t, J = 7.15 Hz, 1H).
6-(4-Methoxybenzyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno-
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one Trifluoroacetate
1
(12). The title compound was prepared using general method B. H
NMR (400 MHz, DMSO-d6) δ 9.22−9.29 (m, 1H), 8.53 (s, 1H),
7.32−7.42 (m, 2H), 6.94 (s, 2H), 5.36 (s, 2H) 4.39 (br s, 2H), 3.75 (s,
3H), 3.51 (br s, 2H), 3.24 (s, 2H). LCMS (MH+, 368.1).
tert-Butyl 5-Oxo-5,6,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine-9(8H)-carboxylate (8). General
method A: Ethyl chloroformate (13.9 mL, 145 mmol) and pyridine
(17.6 mL, 218 mmol) were added to an ice-cooled, stirred mixture of 6
(20 g, 73 mmol) in 1,2-dichloroethane (300 mL). After the addition
was complete, the cooling bath was removed and the mixture was
stirred for 5 h at room temperature. LCMS of the crude mixture
indicated a mixture of the mono- and bis-ethoxycarbonylated products.
The mixture was poured over water (300 mL) and extracted twice
with DCM. The organic layers were combined, dried (MgSO4), and
concentrated under reduced pressure. The residue was diluted with
methanol (300 mL) and water (15 mL) and treated with K2CO3 (5.0
g, 36 mmol). The resulting mixture was stirred at room temperature
for 1 h, during which time the bis-ethoxycarbonylated material was
converted to the desired monoethoxycarbonylated product. The
mixture was poured over water (500 mL), and the desired product was
extracted twice with DCM. The organic layers were combined, dried
(MgSO4), and concentrated under reduced pressure to yield 25 g
(98%) of tert-butyl 3-cyano-2-((ethoxycarbonyl)amino)-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate as a brown solid,
which was used in the following step without further purification.
DIEA (0.29 mL, 1.7 mmol) was added to a mixture of the above
material (1.17 g, 3.33 mmol) and formic hydrazide (240 mg, 4.0
mmol) in DMA (14 mL), and the mixture was heated at 125 °C for 18
h. The mixture was cooled to room temperature and poured over a
mixture of water (150 mL) and brine (100 mL). It was extracted six
times with ethyl acetate, and the combined extracts were dried
(MgSO4) and concentrated. Residual DMA was present, so the
material precipitated from ethyl acetate−hexanes and the resulting
solid was isolated by filtration and air-dried to afford 475 mg (41%) of
8. 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 4.59 (s, 2H), 3.70
6-(4-Chloro-2-fluorobenzyl)-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one tri-
fluoroacetate (13). The title compound was prepared using general
1
method B. H NMR (400 MHz, DMSO-d6) δ 9.19−9.35 (m, 2H),
8.57 (s, 1H), 7.54−7.59 (m, 1H), 7.42−7.51 (m, 1H), 7.23−7.30 (m,
1H), 5.45 (s, 2H), 4.38 (br s, 2H), 3.50 (br s, 2H), 3.23 (t, J = 5.46 Hz,
2H). LCMS (MH+, 390.1).
6-(2-Fluoro-4-methoxybenzyl)-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one Tri-
fluoroacetate (14). The title compound was prepared using general
1
method B. H NMR (400 MHz, DMSO-d6) δ 9.16−9.46 (br s, 2H),
8.56 (s, 1H), 7.34 (t, J = 8.85 Hz, 1H), 6.92 (dd, J = 12.42, 2.38 Hz,
1H), 6.76 (dd, J = 8.66, 2.38 Hz, 1H), 5.38 (s, 2H), 4.38 (s, 2H), 3.77
(s, 3H), 3.45−3.55 (m, 2H), 3.17−3.26 (m, 2H). LCMS (MH+,
386.1).
11,11-Difluoro-6-(4-methoxybenzyl)-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one Hy-
drochloride (15). Compound 9 (346 mg, 0.69 mmol) was diluted in
4N hydrochloric acid in dioxane (5.0 mL, 20 mmol), and the resulting
solution was stirred at room temperature. After 4 h, the mixture was
concentrated under reduced pressure and dried under vacuum to
afford the hydrochloride salt of 15 as a yellow powder. 1H NMR (400
MHz, DMSO-d6) δ 8.56 (s, 1 H), 7.39 (d, J = 8.41 Hz, 2 H), 6.94 (d, J
= 8.41 Hz, 2 H), 5.40 (s, 2 H), 4.49 (br s, 2 H), 4.05 (t, J = 11.42 Hz, 2
H), 3.75 (s, 3 H). LCMS (MH+, 404.1).
6-(4-Chlorobenzyl)-9-methyl-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one
(16a). General method C: Triethylamine (0.034 mL, 0.24 mmol) was
added to a mixture of 10 (100 mg, 0.24 mmol) in 1,2-dichloroethane
(2 mL). After 5 min, aqueous formaldehyde (24 mg, 0.29 mmol) was
G
DOI: 10.1021/acs.jmedchem.7b00302
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Journal of Medicinal Chemistry
Article
added and the resulting mixture was stirred for 1 h. Sodium
triacetoxyborohydride (78 mg, 0.37 mmol) was added, and the
resulting mixture was stirred for 4 h. The mixture was concentrated,
diluted with DMF, filtered, and purified by reverse phase HPLC to
afford the TFA salt of 16a. This material was taken up in DCM,
washed with sodium bicarbonate, dried (MgSO4), and concentrated
under vacuum to afford 11 mg (24%) of 16a as an off-white solid. 1H
NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.38−7.45 (m, 2H), 7.31−
7.37 (m, 2H), 5.38 (s, 2H), 3.67 (s, 2H), 3.18−3.24 (m, 2H), 2.88 (t, J
= 5.69 Hz, 2H), 2.56 (s, 3H). LCMS (MH+, 386.3). Anal.
(C18H16ClN5OS) C, H, N.
6-(4-Chlorobenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-
8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-
c]pyrimidin-5(6H)-one (16h). The title compound was prepared using
general method C. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.40−
7.45 (m, 2H), 7.33−7.38 (m, 2H), 5.39 (s, 2H), 4.00 (dd, J = 11.48,
2.95 Hz, 2H), 3.68 (s, 2H), 3.43 (td, J = 11.73, 1.76 Hz, 2H), 3.13−
3.20 (m, 2H), 2.89 (t, J = 5.71 Hz, 2H), 2.46 (d, J = 7.15 Hz, 2H),
1.79−1.92 (m, 1H), 1.69−1.77 (m, 2H), 1.26−1.40 (m, 2H). LCMS
(MH+, 470.2). Anal. (C23H24ClN5O2S) C, H, N.
6-(4-Chloro-2-fluorobenzyl)-9-((tetrahydro-2H-pyran-4-yl)-
methyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5(6H)-one (16i). The title compound was
prepared using general method C. 1H NMR (400 MHz, DMSO-d6) δ
9.96 (br s, 1H), 8.58 (s,1 H), 7.53−7.61 (m, 1H), 7.43−7.52 (m, 1H),
7.25−7.32 (m, 1H), 5.35−5.58 (m, 2H), 4.69−4.85 (m, 1H), 4.30−
4.46 (m, 1H), 3.76−3.93 (m, 4H), 3.33 (br s, 7H), 2.12 (br s, 1H),
1.67 (d, J = 14.68 Hz, 2H), 1.26 (qd, J = 12.05,4.14 Hz, 2H). LCMS
(MH+, 488.1). Anal. (C23H23ClFN5O2S) C, H, N.
6-(4-Chlorobenzyl)-9-(cyclopropylmethyl)-8,9,10,11-
tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5(6H)-one (16b). The title compound was prepared using
1
general method C. H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H),
7.43 (d, J = 3.18 Hz, 4H), 5.37 (s, 2H), 3.68 (s, 2H), 2.97 (br s, 2H),
2.82−2.87 (m, 2H), 2.40 (d, J = 6.48 Hz, 2H), 0.83−0.96 (m, 1H),
0.49 (d, J = 7.58 Hz, 2H), 0.11 (d, J = 4.52 Hz, 2H). LCMS (MH+,
426.2). Anal. (C21H20ClN5OS) C, H, N.
6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-
8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-
c]pyrimidin-5(6H)-one (16j). The title compound was prepared using
6-(4-Chlorobenzyl)-9-(cyclopropanecarbonyl)-8,9,10,11-
tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5(6H)-one (16c). Piperidine 10 (100 mg, 0.24 mmol),
cyclopropanecarbonyl chloride (0.027 mL, 0.29 mmol), and triethyl-
amine (0.10 mL, 0.73 mmol) were combined in DMA (2 mL) and
stirred for 18 h. The mixture was filtered and purified by prep LCMS
1
general method C. H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H),
7.35−7.33 (m, 2H), 6.91−6.89 (m, 2H), 5.29 (s, 2H), 3.81 (dd, J =
9.0, 2.1 Hz, 2H), 3.72 (s, 3H), 3.60 (br s, 1H), 3.28 (t, J = 9.2 Hz, 2H),
2.95−2.94 (m, 2H), 2.78−2.76 (m, 2H), 2.34 (d, J = 5.8 Hz, 2H),
1.82−1.81 (m, 1H), 1.62−1.59 (m, 2H), 1.12 (qd, J = 10.1, 3.1 Hz,
2H). LCMS (MH+, 466.2). Anal. (C24H27N5O3S) C, H, N.
1
to afford 37 mg (34%) of 16c as an off-white powder. H NMR (400
MHz, DMSO-d6) δ 8.51 (s, 1H), 7.39−7.50 (m, 4H), 5.38 (br s, 2H),
4.64−5.05 (m, 2H), 3.78−4.10 (m, 2H), 2.92−3.15 (m, 2H), 1.94−
2.22 (m, 1H), 0.71−0.80 (m, 4H). LCMS (MH+, 440.2). Anal.
(C21H18ClN5O2S) C, H, N.
6-(2-Fluoro-4-methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)-
methyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5(6H)-one (16k). The title compound was
prepared using general method C. 1H NMR (400 MHz, DMSO-d6) δ
8.50 (s, 1H), 7.30 (t, J = 8.85 Hz, 1H), 6.90 (dd, J = 12.49, 2.32 Hz,
1H), 6.74 (dd, J = 8.60, 2.32 Hz, 1H), 5.34 (s, 2H), 3.80−3.87 (m,
2H), 3.62 (s, 2H) 3.77 (s, 3H), 3.24−3.31 (m, 2H), 2.93−3.02 (m,
2H), 2.76−2.84 (m, 2H), 2.33−2.40 (m, 2H), 1.76−1.91 (m, 1H),
1.57−1.68 (m, 2H), 1.07−1.22 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 161.31 (d, J = 11.0 Hz), 161.42 (d, J = 247 Hz), 154.47,
149.65, 145.12, 144.83, 130.55 (d, J = 5.1 Hz), 129.32, 127.58, 112.48
(d, J = 14.7 Hz), 110.65 (d, J = 2.9 Hz), 110.07, 101.67 (d, J = 25.7
Hz), 67.85, 63.54, 55.65, 51.66, 49.85, 44.92 (d, J = 3.7 Hz), 33.11,
31.62, 25.19. LCMS (MH+, 484.2). Anal. (C24H26FN5O3S) C, H, N.
11,11-Difluoro-6-(4-methoxybenzyl)-9-((tetrahydro-2H-pyran-4-
yl)methyl)-8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5(6H)-one (17). The title compound was
(RS)-9-(2,2-Difluorocyclopropanecarbonyl)-6-(4-methoxybenzyl)-
8,9,10,11-tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-
c]pyrimidin-5(6H)-one (16d). Piperidine 12 (206 mg, 0.43 mmol),
2,2-difluorocyclopropanecarboxylic acid (64 mg, 0.47 mmol), dichloro-
methane (2 mL), triethylamine (0.066 mL, 0.47 mmol), and HOBt
(72 mg, 0.47 mmol) were combined in that order and stirred for 15
min. EDCI (90 mg, 0.47 mmol) was added, and stirring was continued
for 20 h. The mixture was diluted with DCM (10 mL) and then
washed with saturated sodium bicarbonate solution. After filtering
through a phase separating column, the solution was concentrated
under vacuum and the residue was purified by flash LC (elution with
5−75% ethyl acetate in hexanes) to afford a white powder. This
material was lyophilized from 1:1 dioxane−water to afford 27 mg
(13%) of 16d as a white powder. 1H NMR (400 MHz, CDCl3) δ 8.29
(s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 5.50−5.22
(m, 2H), 4.91−4.76 (m,2H), 4.06−3.88 (m, 2H), 3.80 (s, 3H), 3.71 (s,
2H), 3.28 (br s, 2H), 3.19 (br s, 1H), 2.69−2.53 (m,1H), 2.19 (dd, J =
5.0, 12.5 Hz, 1H), 1.80−1.65 (m, 1H). LCMS (MH+, 472.2). Anal.
(C22H19F2N5O3S) C, H, N.
1
prepared using general method C. H NMR (400 MHz, CDCl3) δ
8.42 (s, 1H), 7.41 (d, J = 8.66 Hz, 2H), 6.91 (d, J = 8.66 Hz, 2H), 5.40
(s, 2H), 3.96−4.04 (m, 2H), 3.82 (s, 5H), 3.38−3.47 (m, 2H), 3.28 (t,
J = 11.54 Hz, 2H), 2.58 (d, J = 7.28 Hz, 2H), 1.80−1.94 (m, 1H),
1.69−1.78 (m, 2H), 1.25−1.39 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 160.07, 154.89, 148.47, 145.83, 144.77, 136.13 (t, J = 8.8
Hz), 129.87, 125.08 (t, J = 30.8 Hz), 124.97, 114.82 (t, J = 241 Hz),
114.44, 107.53, 67.71, 62.22, 58.32 (t, J = 28.6 Hz), 55.34, 51.80,
51.40, 32.95, 31.24. LCMS (MH+, 502.2). Anal. (C24H25F2N5O3S) C,
H, N.
PDE Inhibition. PDE1B inhibition IC50 values were determined by
an IMAP assay measuring inhibition of FAM-cAMP hydrolysis by full
length PDE1 enzymes. Specifically, in 1536-well white plates, 250 pg
per well of GST tagged PDE1B was dispensed in 2.5 μL of IMAP assay
buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT,
0.1% fatty acid free BSA, with 10 U/mL calmodulin, and 2.5 mM
CaCl2. Then 30 nL of compound was added from 1 mM stock in
DMSO using the Kalypsys 1536 pintool. Plates were incubated for 5
min at room temperature before dispensing 1.5 μL of 533 nM FAM-
cAMP for a final concentration of 200 nM. The plates were incubated
30 min at room temperature after a brief centrifugation. The assay was
terminated by adding 5 μL of IMAP binding reagent Tb complex to
each well, prepared according to manufacturer’s recommendations.
Plates were incubated 1 h at room temperature and read on the
Viewlux. PDE2A, PDE3A, PDE4D, and PDE10A values were
determined using analogous procedures. PDE5A, PDE6C, PDE7A,
6-(4-Chlorobenzyl)-9-(oxetan-3-yl)-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one
1
(16e). The title compound was prepared using general method C. H
NMR (400 MHz, DMSO-d6) δ 8.50 (s, 1H), 7.39−7.47 (m, 4H), 5.38
(s, 2H), 4.56−4.63 (m, 2H), 4.50 (t, J = 6.05 Hz, 2H), 3.68−3.77 (m,
1H), 3.56 (s, 2H), 2.95−3.03 (m, 2H), 2.69 (t, J = 5.32 Hz, 2H).
LCMS (MH+, 428.1). Anal. (C20H18ClN5O2S) C, H, N.
9-Benzyl-6-(4-chlorobenzyl)-8,9,10,11-tetrahydropyrido-
[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one
1
(16f). The title compound was prepared using general method C. H
NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 7.50−7.42 (m, 6H), 7.41−
7.35 (m, 2H), 7.35−7.30 (m,2H), 5.36 (br s, 2H), 4.39 (s, 4H), 3.57
(br s, 2H), 3.48 (br s, 2H). LCMS (MH+, 462.2). Anal.
(C24H20ClN5OS) C, H, N.
6-(Benzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-
tetrahydropyrido[4′,3′:4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5(6H)-one (16g). The title compound was prepared using
1
general method C. H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H),
7.42−7.29 (m, 5H), 5.37 (s, 2H), 3.81 (dd, J = 2.6, 11.2 Hz, 2H), 3.60
(s, 2H), 3.30−3.22 (m, 2H), 2.97 (br s, 2H), 2.79 (t, J = 5.5 Hz, 2H),
2.39−2.31 (m, 2H), 1.82 (br s, 1H), 1.61 (d, J = 11.9 Hz, 2H), 1.19−
1.05 (m, 2H). LCMS (MH+, 436.0). Anal. (C23H24ClN5O2S) C, H, N.
H
DOI: 10.1021/acs.jmedchem.7b00302
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Journal of Medicinal Chemistry
Article
PDE8A, PDE9A, and PDE11A values were determined at BPS
Bioscience.
with a reversed-phase column (Thermo Hypersil GOLD 3 μm drop-in
guard cartridges, 10 mm × 2.1 mm). Mobile phase was consisted of
0.1% formic acid in water and of 0.1% formic acid in ACN with a flow
rate of 0.7 mL/min. Eluent was directed to an API4000 triple
quadruple mass spectrometer (AB Sciex, Framingham, MA) equipped
with a turbo electrospray interface. Multiple reaction monitoring
(MRM) transition in positive ion mode was used. Integration of the
sample peaks was performed using AB Sciex Analyst and
DiscoveryQuant software; peak area ratio of the analyte to the
internal standard was determined accordingly. In vitro intrinsic
clearance (CLint) was calculated by dividing the first-order degradation
rate constant (Kdeg) by the concentration of microsomal protein used
in the incubation. Kdeg was the slope using least-squares fit to the curve
of percent test compound remaining vs time (0−30 min for RLM and
0−60 min for HLM).
Protein Expression, Purification, Crystallization, and Crystal
Handling. A polyhistidine-tag−tobacco etch virus (TEV) protease
cleavage site sequence followed by the bacterial codon optimized
cDNA corresponding to human PDE1B1 (UniProt entry Q01064-1)
residues 146−506 and PDE10A1 (UniProt entry Q9Y233-1) residues
439−779 were subcloned into separate pNIC28-Bsa4 expression
vectors. The pNIC28-Bsa4 was a gift from Opher Gileadi (Addgene
plasmid no. 26103).38 The resulting plasmids were transformed into
the Escherichia coli Rosetta 2 (DE3) strain (EMD Millipore). Cultures
grown in Terrific Broth medium were induced with 0.1 and 0.5 mM
isopropyl β-D-thiogalactopyranoside (IPTG) at OD600 = 3.0 for
PDE1B and PDE10A, respectively. At the time of induction, the
incubation temperature was lowered to 18 °C. The cells were
harvested 16 h post induction.
Rat Pharmacokinetics Determination. Pharmacokinetics prop-
erties in male SD rats (300−350g) were determined following
intravenous (IV) and oral (PO) administration. For IV dosing (N = 3,
1 mg/kg), rats were catheterized in jugular and femoral vein. For PO
dosing (N = 3, 5 mg/kg), rats were catheterized in femoral vein. Test
article was formulated in NMP:PEG400:water (10:40:50). Rats were
not fasted during this study. Blood was sampled at 0 (predose), 0.033,
0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 16, and 24 h following IV and at 0
(predose), 0.25, 0.5, 1, 2, 4, 8, 12, 16, and 24 h following PO dosing.
Plasma was isolated by centrifugation, and all samples were frozen at
−80 °C. Calibration standards were prepared by the addition of
known concentrations of test article to blank rat plasma to provide a
calibration range of 0.5−2000 ng/mL. Then 50 μL plasma samples or
calibration standard was added to 250 μL of internal standard solution
in acetonitrile. Samples were vortex mixed and centrifuged at 12000
rpm for 5 min at 4 °C. Supernatant (100 μL) was transferred to
labeled autosampler vials containing 300 μL of mobile phase (water/
acetonitrile/formic acid, 90/10/0.2%), vortex mixed, and analyzed by
LC-MS/MS. A bioanalytical method was developed for the
quantification of test article in rat plasma. Method development and
sample analysis was conducted using a Waters Quattro Premier LC-
MS/MS system equipped with Waters Acquity UPLC system. Then 5
μL of the samples were analyzed using a Waters Acquity UPLC system
equipped with a C 18 reversed-phase column (Phenomenex Kinetex
C18, 1.7 μm, 2.1 mm × 50.0 mm). Mobile phase consisted of 0.1%
formic acid in water and of 0.1% formic acid in acetonitrile with a flow
rate of 0.7 mL/min. Eluent was directed to a Waters Quattro Premier
mass spectrometer equipped with a turbo electrospray interface.
Multiple reaction monitoring (MRM) transition in positive ion mode
was used.
For CNS penetration studies male SD rats (300−350 g) were dosed
PO (N = 3, 10 mg/kg). Test article was formulated in NMP:PEG400:-
water (10:40:50). Animals were anesthetized, and approximately 0.3
mL of blood from each rat was collected via cardiac puncture into
tubes containing lithium heparin as the anticoagulant at 1 h post dose.
Plasma was isolated by centrifugation. Animals were then decapitated
and brains were removed. All samples were stored frozen at
approximately −80 °C until analysis. Plasma analysis was performed
as described above.
One hemisphere of each brain sample was weighed. One to five
dilutions (w/v) were prepared by addition of 1:1 mixture of water/2-
propanol (4-fold of the brain weight) to each brain sample. Brains
were homogenized by Fast Prep Calibration standards (ranging from
0.500 to 2000 ng/mL), quality control samples, and test samples
containing the internal standard (IS) were separated from rat brain
preparations via protein precipitation by addition of 100 μL of 75:25
acetonitrile/water solvent containing analyte calibrants and 300 μL of
acetonitrile/IS solution mix (40 ng/mL in acetonitrile) to 100 μL
brain homogenate. Following vortex mixing and centrifugation, 20 μL
aliquots of the supernatants were diluted with 580 μL of 20:80
acetonitrile/water containing 0.2% formic acid and mixed for the 10
mg/kg study. Following vortex mixing and centrifugation, 100 μL
aliquots of the supernatants were diluted with 300 μL of 10:90
acetonitrile/water containing 0.2% formic acid and mixed for the 1.0
mg/kg study.
Following cell lysis, the PDE1B protein was affinity purified with
Ni-NTA resin. Following TEV protease treatment, and concurrent
dialysis, the protein was passed over a bed of Ni-NTA resin. The flow-
through was concentrated and injected onto a HiLoad 16/600
Superdex 200 column equilibrated with 10 mM Tris, 100 mM NaCl,
and 1 mM TCEP, pH 8.0. The elution peak corresponding to the
target protein was concentrated to 11 mg/mL. The protein was
incubated with 200 μM BAY 60-7550 and crystallized by the hanging
drop vapor diffusion method at 20 °C with a well solution containing
100 mM HEPES pH 6.9 and 3.1 M NaCl.39 Compound 3 and 16j
were introduced by soaking crystals overnight in solutions matching
the well solution but with 25% glycerol and 2.5 mM compound.
Crystals were mounted in cryo loops and flash-frozen in liquid
nitrogen.
The PDE10A protein was purified as previously described.40 The
purified protein in 20 mM HEPES, 150 mM NaCl, 5 mM DTT, pH
7.5, was concentrated to 15 mg/mL. Apo crystals of human PDE10A
were obtained by the hanging drop vapor diffusion method at 20 °C
with a well solution containing 15% PEG3350, 200 mM calcium
acetate, and 10 mM 2-mercaptoethanol. Crystals were first soaked in
well solution containing 1 mM 3-isobutyl-1-methylxanthine (IBMX)
overnight. Compound 16d was introduced through a second soaking
step by transferring the IBMX soaked crystals to a solution matching
the well solution supplied with 5 mM of compound 16d. After 24 h,
crystals were washed with a cryo solution (75% well solution plus 25%
ethylene glycol) and flash-frozen in liquid nitrogen.
Structure Determination. Single-crystal X-ray diffraction data
were collected at cryogenic temperature (100 K). The PDE1B data
were collected at beamline 5.0.2 at the Advanced Light Source,
Lawrence Berkeley National Laboratory (Berkeley, CA) and processed
with DIALS.41 The PDE10A data were collected at the Stanford
Synchrotron Radiation Lightsource (SSRL), SLAC National Accel-
erator Laboratory (Menlo Park, CA) and processed with XDS.42 The
initial phases were generated by molecular replacement using
publically available PDE1B and PDE10A catalytic domain structures
(PDBs 1TAZ and 5C2H, respectively).26,43 The structures were built
with COOT and refined with Phenix.44,45 The X-ray data processing
and structure refinement statistics are reported in Supporting
Information, Table S1. Figures were generated with PyMOL.46 Only
chain A of the PDE10A−16d cocomplex structure was used for ligand
binding analysis. In chain B, the ligand conformation is influenced by
crystal packing (Supporting Information, Figure S2).
Metabolic Stability in the Presence of Liver Microsomes.
Test compound was incubated at a final concentration of 1 μM in a
reaction mixture containing human or rat liver microsomes of 0.5 mg
protein/mL in Tris buffer at 37 °C. The reaction was started by
addition of NADPH (1 μM). Aliquots of the incubation mixture were
removed and quenched with three volumes of cold ACN containing
internal standard, indomethacin (0.5 μM) at 0, 5, 15, and 30 min for
RLM and at 0, 15, 30, and 60 min for HLM. The samples were vortex
mixed and centrifuged at 4000 rpm for 10 min at 40 °C, and the
supernatant was transferred to a 384-well plate followed by dilution
with same volume of distilled water. Then 5 μL of the samples were
analyzed using a Shimadzu HPLC system (Kyoto, Japan) equipped
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Article
Then 5 μL of each sample was applied to a Kinetex 1.7 μm C18,
100 Å, 50 mm × 2.1 mm column (Phenomenex, Torrance, CA) and
eluted with a mobile phase gradient (initial 10% B hold for 0.1 min,
then 10% B to 90% B in 1.0 min) consisting of solution A (0.1%
formic acid in water) and solution B (0.1% formic acid in acetonitrile).
The column temperature was maintained at 30 °C. The column eluent
was subjected to positive-mode electrospray ionization (ES+), and the
analytes were detected with a QTrap 5500 quadrupole/ion trap mass
spectrometer (AB Sciex, Framingham, MA).
Novel Object Recognition Training and Testing. Rats were
handled for three days and habituated to the testing arena for 10 min.
On training day, drug was administered orally in a vehicle of 10%
DMSO, 30% PEG 400, and 60% saline at a volume of 2 mL/kg.
Animals were then placed into the training box and allowed to explore
two identical objects for either 3 min (to induce a weak memory) or
for 15 min (to induce a strong memory). Memory retention was tested
for 5 min 24 h after training. Animals were presented with one
“familiar” (training) and one “novel” object. Objects were placed into
the central area of the box, and the spatial position of objects (left−
right sides) was counterbalanced between subjects. After each
experimental subject, the apparatus and the objects were thoroughly
cleaned with 90% ethanol, dried, and ventilated for a few minutes. To
determine memory, an object-discrimination index (DI) was
determined as [novel exploration − familiar exploration]/ [total
exploration], which is shown as the memory score (DI × 100). In
addition, the time exploring the objects during training was recorded.
Data were analyzed by least-squares means planned comparisons, the
results of which are presented. All data are presented as the mean
SEM.
Science, Office of Basic Energy Sciences under Contract No.
DE-AC02-76SF00515. The SSRL Structural Molecular Biology
Program is supported by the DOE Office of Biological and
Environmental Research and by the National Institutes of
Health, National Institute of General Medical Sciences
(including P41GM103393). The contents of this publication
are solely the responsibility of the authors and do not
necessarily represent the official views of NIGMS or NIH.
We thank Kristen Arienti for proof reading the manuscript and
Grace Yan for additional pharmacological experiments.
ABBREVIATIONS USED
■
br s, broad signal; CREB, CAMP-response element binding
protein; DIEA, diisopropylethylamine; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; FAM-CAMP, FAM-
cAMP, 2′-(6-[fluoresceneinyl] aminohexylcarbamoyl)-
adenosine-3′,5′-cyclic monophosphate; HLM, human liver
microsomes; IMAP, immobilized metal ion affinity-based
fluorescence polarization; HOBt, 1-hydroxybenzotriazole;
MDCK, Madin−Darby canine kidney; NOR, novel object
recognition; RLM, rat liver microsomes
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ASSOCIATED CONTENT
* Supporting Information
̈
■
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The Supporting Information is available free of charge on the
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The crystal structures of 3 in PDE1B (5UP0), 16d in PDE10A
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Corresponding Author
*Phone: 858-736-3044. E-mail: bdyck@dartneuroscience.com.
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Laurent Gomez: 0000-0002-9172-7840
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