Design and Synthesis of Selective Phosphodiesterase 4D (PDE4D) Allosteric Inhibitors for the Treatment of Fragile X Syndrome and Other Brain Disorders

Article abstract of DOI:10.1021/acs.jmedchem.9b00193

Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site f

Full text of DOI:10.1021/acs.jmedchem.9b00193

Drug Annotation
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Design and Synthesis of Selective Phosphodiesterase 4D (PDE4D)
Allosteric Inhibitors for the Treatment of Fragile X Syndrome and
Other Brain Disorders
Mark E. Gurney,*,† Richard A. Nugent,† Xuesheng Mo,† Janice A. Sindac,† Timothy J. Hagen,‡
David Fox, III,§,+ James M. O’Donnell,∥ Chong Zhang,∥,□ Ying Xu,∥ Han-Ting Zhang,⊥
Vincent E. Groppi,# Marc Bailie,∇ Ronald E. White,○ Donna L. Romero,◆ A. Samuel Vellekoop,¶
Joel R. Walker,¶ Matthew D. Surman,¶ Lei Zhu,¶ and Robert F. Campbell¶,●
†Tetra Discovery Partners, Inc., 38 Fulton Street West, Grand Rapids, Michigan 49503, United States
‡Department of Chemistry and Biochemistry, Northern Illinois University, 1425 West Lincoln Highway, DeKalb, Illinois 60115,
United States
§Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, Washington 98110, United States
∥Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State
University of New York, Buffalo, New York 14214-8033, United States
⊥Departments of Behavioral Medicine & Psychiatry and Physiology, Pharmacology & Neuroscience, Rockefeller Neurosciences
Institute, West Virginia University Health Sciences Center, 1 Medical Center Drive, Morgantown, West Virginia 26506, United
States
#Michigan Drug Discovery, Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48103,
United States
∇INDS Inc., 6111 Jackson Road, Suite 100, Ann Arbor, Michigan 48103, United States
○White Global Pharma Consultants, 31 Kinglet Drive, South Cranbury, New Jersey 08512, United States
◆Pharma-Vation Consulting, LLC, 1201 Turnberry Ridge Court, Chesterfield, Missouri 63005, United States
¶Albany Molecular Research, Inc., 21 Corporate Circle, Albany, New York 12203, United States
S
* Supporting Information
ABSTRACT: Novel pyridine- and pyrimidine-based allosteric
inhibitors are reported that achieve PDE4D subtype selectivity
through recognition of a single amino acid difference on a key
regulatory domain, known as UCR2, that opens and closes over the
catalytic site for cAMP hydrolysis. The design and optimization of
lead compounds was based on iterative analysis of X-ray crystal
structures combined with metabolite identification. Selectivity for
the activated, dimeric form of PDE4D provided potent memory
enhancing effects in a mouse model of novel object recognition with
improved tolerability and reduced vascular toxicity over earlier
PDE4 inhibitors that lack subtype selectivity. The lead compound,
28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.
dimeric isoforms is increased in response to cAMP signaling
through phosphorylation of UCR1 by protein kinase A
(PKA).9−12
The importance of the enzyme for normal brain function is
underscored by the discovery of PDE4D mutations in an
ultrarare, neurodevelopmental disorder known as acrodysostosis
type 1 with or without hormone resistance (ACRDYS2), a
condition in which brachydactyly (short fingers and toes) is
INTRODUCTION
■
The phosphodiesterase-4 (PDE4) enzymes regulate the spatial
and temporal patterning of signaling through the cAMP second
messenger system by hydrolysis of the cyclic nucleotide.1−4 The
PDE4 gene family consists of four subtypes, PDE4A-D, that are
distinguished from other PDE gene families by the presence of
upstream conserved regions known as UCR1 and UCR2.5,6
PDE4 enzymes can be present in cells as either dimers or
monomers. UCR1 is required for dimerization while UCR2
regulates enzyme activity by opening and closing over the active
site and thereby controls access of cAMP.7,8 The basal activity of
Received: January 30, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.9b00193
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Journal of Medicinal Chemistry
Drug Annotation
Figure 1. (1) Burgin et al. compound D159687;7 (2) Naganuma et al. compound 33;49 (3) Hagen et al. compound 8.50 The aliphatic substituents on
the core project into a pocket of the active site known as the Q region.51 A pair of aromatic arms projects from the core. These have been labeled Ar1
and Ar2 as in Burgin et al.7
accompanied by intellectual disability with affected individuals
showing reduction in IQ.13−17 The ACRDYS2 mutations are
exclusively missense mutations of residues on the surface of the
protein. In one patient, the UCR1 PKA phosphorylation site is
mutated from serine to alanine, thereby preventing PKA
activation in response to cAMP signaling.15,18 A second subset
of ACRDYS2 mutations alter contact residues between UCR2
and the catalytic domain, thereby affecting the closure of UCR2
across the active site.19 The remaining ACRDYS2 mutations
occur at the base of the four-helix bundle needed for
dimerization and appear to elevate basal enzyme activity.20
Genetic variation in PDE4D also contributes to biological
variation in human cognitive ability. The PDE4D gene is a
consistent hit across multiple, large-scale, genome-wide
association studies (GWAS) of human cognition.21−24 Because
of the large size of the gene, more than 1 million base pairs, these
studies associate allelic variation over 5′ exons encoding dimeric
forms of the PDE4D enzyme with human cognitive ability.25
Thus, as with the ultrarare ACRDYS2 mutations, common
genetic variants identified by GWAS also show the importance
of dimeric isoforms of PDE4D for normal brain function.
The role in cognition of second messenger signaling through
cAMP has been studied in model organisms. Dunce, the first
mutation shown to affect learning and memory in the Drosophila
fruit fly, is a deletion of the single PDE4 gene present in
flies.26−28 Deletion of PDE4 impairs associative memory by
altering the spatial and temporal patterning of cAMP signal-
ing.4,29 Rutabaga, which also impairs associative memory in the
fruit fly, is a mutation of the calcium-calmodulin adenylate
cyclase.30 Studies in genetically engineered mice place PDE4D
downstream of calcium influx through the N-methyl D-aspartate
receptor (NMDA-R) with consequent activation of calcium/
calmodulin adenylate cyclase in a pathway important for early-
and late-stage memory formation as well as hippocampal
neurogenesis and the expression of brain-derived neurotrophic
factor (BDNF).31−36
We showed previously that it is possible to design subtype-
selective, PDE4D allosteric inhibitors with improved tolerability
and reduced vascular toxicity.7 Subtype selective PDE4D
allosteric inhibitors exploit a binding pose in which the inhibitor
captures a regulatory helix from UCR2 in trans- across the
opposite active site.7,8 As we describe below, such compounds
are more potent against dimeric as compared to monomeric
forms of PDE4D and can be optimized for potency against the
activated as compared to the basal state of the PDE4D dimer.
This pharmacological profile contrasts with the profiles of
competitive, active-site directed PDE4 inhibitors.
RESULTS
■
PDE4 allosteric inhibitors utilize a common pharmacophore in
which an aromatic core forms a hydrogen bond with an
invariant, active site glutamine and a pair of aromatic arms
capture UCR2 in trans- across the active site thereby blocking
access by cAMP.7,48−50 Within UCR2, a single amino acid
difference of a phenylalanine in PDE4D (Phe271) and a tyrosine
in PDE4A-C can be exploited for subtype selectivity.7 Selective
PDE4B inhibitors also fit this pharmacophore,49 however,
PDE4B allosteric inhibitors capture a different, C-terminal
regulatory helix (CR3) in cis- across the active site.48 The initial
series of PDE4D allosteric inhibitors that we described used a
methoxyphenyl core in which the oxygen forms a hydrogen
bond with the invariant glutamine (1, Figure 1).7 We speculated
that the pyrimidine core of the series of PDE4B inhibitors
reported by Naganuma (2)49 could be optimized to capture
UCR2.
The choice of the in vitro assay format for measuring
inhibition of PDE4 enzymatic activity is critical. We utilized a
real-time, coupled enzyme assay in which hydrolysis of cAMP to
AMP leads to the oxidation of NADH which can be measured
spectroscopically.7 Many high-throughput assays of PDE4
enzymatic activity, in contrast, use a fluorescein-labeled cAMP
substrate that when hydrolyzed allows detection of AMP using
fluorescence polarization. However, the fluorescein label
prevents closure of UCR2 and thereby alters the kinetics of
PDE4 allosteric inhibitors (Supporting Information, Figure 1).
PDE4 enzymes are present in cells as dimers (if they contain
UCR1) or as monomers (if they lack UCR1).52,53 The basal
activity of dimeric forms of PDE4 is increased by phosphor-
ylation of a serine in UCR1 by PKA.10,12 Mutating the serine to
aspartic acid mimics PKA phosphorylation and increases the
activity of the enzyme. In the case of PDE4D7, we measured an
8-fold increase in hydrolytic activity due to mutation of
serine129 to aspartic acid (notation: S129D). For screening,
we used mutationally activated, dimeric forms of the enzymes,
Multiple studies show the benefit of PDE4 inhibition in
mouse models of learning and memory, major depression, and
neurodegeneration.37−42 Although several PDE4 inhibitors are
used clinically, the effective dose is limited by undesirable gastro-
intestinal side effects such as nausea, vomiting, and diar-
rhea.43−45 In rodents and other species, PDE4 inhibitors may
also cause vascular inflammation, particularly in the gut,
however, that toxicity has not been documented in humans.46,47
Marketed PDE4 inhibitors such as roflumilast and apremilast
lack PDE4 subtype selectivity, lack selectivity for dimeric as
compared to monomeric isoforms of the PDE4 enzymes, and
inhibit equally the activated and basal states.
B
DOI: 10.1021/acs.jmedchem.9b00193
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Journal of Medicinal Chemistry
Drug Annotation
Table 1. Inhibitory Activity of Triazine Ar1 Substitution
a
b
c
d
e
compd
R
PDE4D7-S129D IC50 (nM) PDE4B1-S133D IC50 (nM) PDE4D7-S129(wt) IC50 (nM) ratio B/D ratio basal/activated
4
5
6
7
8
9
10
11
−(CH2)3-OH
−CH2CH2OH
−CH2CONH2
−CH2OH
−CONH2
−CH(Me)CO2H
−CO2H
0.2
0.5
5
12
22
96
213
681
11
24
48
105
44
1090
241
781
27
55
48
10
9
2
11
1
135
204
41
30
11
102
203
351
252
NC
482
f
2
6
−CH2CO2H
4040
1
a
b
PDE4D7-S129D is a dimeric isoform of PDE4D that contains a UCR1 mutation (S129D) that mimics PKA phosphorylation. PDE4B1-S133D is
a dimeric isoform of PDE4B that also contains a UCR1 mutation (S133D) that mimics PKA phosphorylation. PDE4D7-S129(wt) is the native
isoform of PDE4D7 that lacks the UCR1 phosphorylation mimetic mutation. B/D is the ratio of IC50 for PDE4B1-S133D/PDE4D7-S129D.
Basal/Activated is the ratio of IC50 for PDE4D7-S129(wt)/PDE4D7-S129D. NC -Not calculated as IC50 > 10,000 nM. See experimental section
c
d
e
f
for methods and detailed synthetic procedures.
Figure 2. PDE4D allosteric binding site. (A) view of a PDE4 dimer (PDB 4WZI) showing the four-helix bundle and UCR2 from one subunit (colored
red) positioned in trans- across the active site of the opposite subunit (colored blue).8 (B) Views of compounds 5 (PDB 6NJI) and 6 (PDB 6NJH)
bound in the PDE4D allosteric site showing key polar contacts. Magnesium (green sphere), zinc (purple sphere), water (red spheres), UCR2 (cyan),
and catalytic domain amino acid side chains (green).
PDE4D7-S129D and PDE4B1-S133D. These were produced
using a baculovirus expression system in Sf9 cells and purified
using C-terminal affinity tags and column chromatography.
Our initial study of the structure−activity relationship (SAR)
in a triazine series as in 3 confirmed that Ar1 and Ar2 could be
tuned for subtype selectivity.50 Replacement of the Ar2
chlorothiophene in 2 with the 3-chlorophenyl in 1, consistently
provided compounds with PDE4D selectivity through capture
of UCR2 and not CR3. Exploration of Ar1 replacements
achieved a 3000-fold range in PDE4D potency across a series of
amides or alcohols with the primary alcohols, providing the
greatest potency and selectivity (Table 1). Co-crystal structures
of ethyl alcohol (5) and amide (6) derivatives show both
inhibitors hydrogen bond to the invariant glutamine671 (Q671)
in the active site through the triazine core. A second hydrogen
bond is observed from the amine linker to a stable water
molecule near the metal center and tyrosine461 (Y461) of the
catalytic domain. In addition, the cocrystal structure revealed
that the amide nitrogen in 6 participates in a hydrogen bond
through a water network with UCR2 threonine275 (T275),
while the oxygen hydrogen bonds with the catalytic domain
through a water molecule that coordinates to both histidine506
(H506) and the first shell of waters surrounding the neighboring
magnesium atom. The alcohol 5 lacks the interaction with
UCR2 while maintaining the interaction with the catalytic
domain. Phenylalanine271 (F271), which projects from UCR2
into the allosteric site, is the key residue for PDE4D selectivity.
In the UCR2 of PDE4A−C, the phenylalanine is replaced by a
tyrosine. When 5 or 6 occupy the allosteric site, UCR2 closes
across the binding site and accommodates the phenylalanine but
does not tolerate the protruding para-hydroxyl substituent of a
tyrosine (Figure 2, see also Figure 7). Although potent and
selective, the triazines were abandoned as they featured low or
negligible solubility, poor cellular permeability, and high plasma
protein binding.
Published cocrystal structures with 2 and PDE4B showed that
the methyl and ethyl arms extending from the pyrimidine core
project into an invariant hydrophobic pocket known as the Q-
region.7,48 The Q-region provides an opportunity to adjust the
potency, as long as groups projecting into the pocket are kept
small. We explored a fused cyclopentyl group as a conservative
replacement for the 6-methyl and 5-ethyl substituents of 2. Like
C
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Journal of Medicinal Chemistry
Drug Annotation
a
Table 2. Inhibitory Activity of Cyclopentylpyridine Ar1 Substitution
compd
R
PDE4D7-S129D IC50 (nM) PDE4B1-S133D IC50 (nM) PDE4D7-S129(wt) IC50 (nM) ratio B/D ratio basal/activated
12
13
14
15
16
17
4-CH2CH2OH
4-(CH2)2CONH2
4-CH2CO-NMe2
4-CH2CO-N(H)Me
3- CH2CH2OH
4-CH2CO2H
9
35
51
133
226
1165
562
166
356
98
1419
1642
2111
1283
914
60
5
7
1
6
226
37
18
15
28
1991
6325
b
NT
1
a
b
See Table 1 for assay definitions. See experimental section for methods and detailed synthetic procedures. NT: not tested.
Figure 3. Timeline plot for chemical optimization of different cores and Q region substituents. Compound symbols are colored by linker type, and
inhibitory potency against PDE4D-S129D is indicated by the size of the symbol.
a
Table 3. Inhibitory Activity of Linker Modified Analogues
PDE4D7-S129D IC50
(nM)
PDE4B1-S133D IC50
(nM)
PDE4D7-S129(wt) IC50
(nM)
ratio
B/D
ratio
basal/activated
compd series
L
R
18
19
20
21
22
23
24
25
26
27
A
A
A
A
B
B
B
B
B
B
CH2
NH
SO2
N(Me)
NH
CH2
O
CH2
NH
O
−CH2CONH2
−CH2CONH2
−CH2CONH2
−CH2CONH2
−CH2CH2OH
−CH2CONH2
−CH2CH2OH
−CH2CH2OH
−CH2CONH2
−CH2CONH2
8.6
42.1
475
733
1.1
1.7
1.8
2
137
136
3830
3287
57
225
58
584
43
810
16
3
8
94
40
1665
b
NT
NT
b
4
149
313
251
534
146
52
132
32
292
17
27
135
184
139
267
56
2.6
35
953
2431
69
a
b
See Table 1 for assay definitions. See experimental section for methods and detailed synthetic procedures. NT: not tested.
D
DOI: 10.1021/acs.jmedchem.9b00193
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Drug Annotation
Figure 4. Effect of amine and methylene linker replacements on PDE4 selectivity across multiple cores. The ratios of selectivity for PDE4B3-S133D/
PDE4D7-S129D are plotted versus PDE4D7-S129(wt)/PDE4D7-S129D. The dotted line is the best-fit in the methylene linker series (Bravais
Pearson test, r = 0.677) for direct proportionality between selectivity for PDE4B-S133D versus PDE4D7(wt). Symbols: blue solid circles, methylene
linker; yellow solid circles, amine linker; red star, apremilast; red solid triangle, rolipram. The relative size of the symbols indicates potency against
PDE4D7-S129D. Units are in nM.
Figure 5. Comparison of methylene (compound 23, magenta) and amine (compound 6, yellow) linkers. (A) Only residues from the compound 6
cocrystal structure are shown for simplicity as no major changes are observed in the side chain rotamers. Waters associated with 6 (red) and 23 (blue)
are shown with hydrogen bonds for 6 (black) and 23 (red). UCR2 is in cyan and the zinc atom is a gray sphere. (B) Overlay of 6 (yellow) and 23
(magenta) with 2Fo − Fc electron density contoured at 1σ (blue mesh) and Fo − Fc electron density contoured at 3σ (green/red mesh). Waters
associated with 6 (red) and 23 (sand) are shown with the hydrogen bond unique to 6 highlighted in red.
the triazine series, the Ar2 chlorothiophene was replaced with
the 3-chlorophenyl. We found that the SAR for Ar1 could be
transferred to the new series with alcohols and amides showing
the best PDE4D potency and selectivity (Table 2). Exploring
meta- versus para- substitution (12 vs 16) of the Ar1 phenyl
showed that the trajectory of the distal alcohol or amide was
important for potency, further supporting the conclusions drawn
from the structural studies of the triazine series. However, lead
compounds had low solubility with high P-glycoprotein efflux in
MDCK-MDR1 cells and therefore were abandoned. SAR
studies then focused on 6-substituted pyridines or pyrimidines.
A timeline plot of the different cores that were explored over the
course of the lead optimization effort is shown in Figure 3.
A survey of linkers using the cyclopentylpyridine core and an
Ar1 amide revealed that a methylene linker was preferred over an
amine linker for PDE4D potency and selectivity (Table 3). The
6-ethylpyrimidine series confirmed that trend when matched
pairs of Ar1 amides and alcohols were compared. This affects
PDE4 subtype selectivity as shown in Figure 4 for compounds in
which Ar2 is fixed as 3-chlorophenyl and the linker is either a
methylene or an amine while the core varies. In addition,
compounds with a methylene linker show good proportionality
between PDE4B/PDE4D and basal/activated selectivity.
Apremilast, an active site inhibitor competitive with cAMP has
no selectivity for PDE4B versus PDE4D, nor does it show
selectivity for the activated enzyme. (R)-rolipram, a prototypical
E
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Journal of Medicinal Chemistry
Drug Annotation
Figure 6. Mouse NOR and anesthesia duration test. (A) 23 significantly improved novel object discrimination in comparison to vehicle (VEH; Two-
Way ANOVA F(4,28) = 3.96, p < 0.001) at doses of 1 and 3 mg/kg. (B) 28 significantly improved novel object discrimination (F(6,42) = 4.18, p < 0.005) at
doses above 0.3 mg/kg PO. (C) 29 significantly improved novel object discrimination (F(5,35) = 8.26, p < 0.005) at doses above 3 mg/kg PO as did
rolipram (ROL) at a dose of 0.1 mg/kg SC. (D) 23 significantly reduced the duration of anesthesia induced by ketamine and xylazine, a
pharmacological construct of emetic potential (F(3,27) = 20.9, p < 0.001) at doses of 10 and 30 mg/kg PO. (E) 28 had no significant effect on anesthesia
duration at doses of 3, 10, and 30 mg/kg PO (F(4,36) = 1.08), while (F) 29 significantly reduced anesthesia duration at doses of 3 mg/kg PO and higher
(F(4,28) = 4.7, p < 0.005). Rolipram significantly reduced anesthesia duration at 0.1 mg/kg IP. P-values shown are ns, not significant; * p < 0.01, ** p <
0.05, and *** p < 0.001 (Dunnett’s multiple comparison test).
allosteric inhibitor, is 5.5-fold selective for PDE4D versus
PDE4B and 21-fold selective for the activated form of PDE4D7
(Figure 4).
PDE4D versus the basal enzyme.54 We postulated that as we
improved PDE4D subtype selectivity, as well as the window
between inhibition of the activated and basal forms of the
PDE4D dimer, we would improve tolerability and reduce
toxicity.
To profile in vivo pharmacology, compounds were screened
for improvement of long-term memory in mice using novel
object recognition after a 24 h delay and then counter screened
for emetic potential (Figure 6). As mice are unable to vomit, a
pharmacologic construct of PDE4 inhibitor-induced emesis was
used to assess potential tolerability, the shortening of the
duration of anesthesia induced by the combination of ketamine
and xylazine.55,56 The anesthesia duration test provides an
estimate of the potential therapeutic window between efficacy
and tolerability.
Compound 23 (Table 3), an early lead, was moderately stable
in human, dog, mouse, rat, and monkey liver microsomes (54−
90% parent remaining at 30 min), with good permeability and
low efflux in MDR1-MDCK cells (A−B = 24.1 × 10−6 cm/s; B−
A = 22 × 10−6 cm/s; efflux ratio = 0.9). Aqueous kinetic
solubility was low (<1.6 μM at pH 7), while solubility increased
(100 μM) in fasted state simulated intestinal fluid (FaSSIF).
Protein binding in plasma from humans, dogs, rats, and mice was
>99.5−99.8%, although there was no shift in the IC50 for
PDE4D7-S129D inhibition in the presence of bovine serum
albumin (BSA) at 1 mg/mL. 23 was a moderate cytochrome
P450 inhibitor (CYP3A4 IC50 = 5.9 μM, 2C19 = 29 μM; 2D6 >
100 μM; 2C9 > 100 μM). Evaluation of the compound in mice
showed good oral bioavailability when dosed in 5% DMSO, 5%
Solutol, 90% PBS (F% = 53%), extended absorption over 4−8 h,
and excellent distribution to brain (brain/plasma ratio = 2.7
based on AUC). However, half-lives in plasma and brain were
short (t1/2 = 0.46 and 0.44 h, respectively). 23 improved
discrimination of the novel object at oral doses of 1 and 3 mg/kg,
while it reduced anesthesia duration at doses of 10 and 30 mg/
Compounds with the methylene linker tend to achieve greater
PDE4D selectivity than those with the amine linker. Comparing
PDE4D cocrystal structures of compounds with a methylene
linker with those containing an amine linker shows little
movement of the core which is fixed in place by the hydrogen
bond to the invariant active site glutamine671 (Q671) and the
phenylalanine−isoleucine clamp.51 Also, both 6 and 23 bind
similarly to UCR2 threonine275 (T275). However, the
methylene linker in 23, unlike the amine linker in 6, is unable
to hydrogen bond with the water molecule coordinated between
catalytic domain residues tyrosine461 (Y461), aspartic acid620
(D620), and a water molecule near the zinc atom (Figure 5).
This has the effect of reducing binding affinity for the catalytic
domain while maintaining UCR2 interactions, effectively
improving selectivity for the PDE4D subtype.
On the basis of the genetic analysis of ACRDYS2 mutations,
our therapeutic hypothesis was that inhibition of the activated,
dimeric forms of PDE4D would be associated with cognitive
benefit. We noted that as compounds achieved greater PDE4D
subtype selectivity, selectivity for the activated form of the
PDE4D dimer also increased (Figure 4). This is most striking for
the compounds with a methylene linker where there is a good
correlation between PDE4B/PDE4D selectivity and basal/
activated selectivity. In contrast to the allosteric inhibitors,
PDE4 inhibitors such as apremilast (30) that bind in the active
site competitively with cAMP have no PDE4 subtype selectivity
due to the absolute amino acid sequence conservation of the
active site,51 nor are they selective for the activated versus basal
forms of the PDE4 enzymes (Figure 4). (R)-Rolipram, a
prototypical allosteric inhibitor that is highly efficacious in many
preclinical models, but also highly emetic, has 5.5-fold selectivity
for PDE4D versus PDE4B and 21-fold selectivity for activated
F
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Journal of Medicinal Chemistry
Drug Annotation
a
Table 4. In Vitro Metabolism of Compound 23 by Human and Rat Hepatocytes
metabolites
M1
M2
M3
M4
M5
M6
367
parent
366
[M + H]+,m/z
ΔMW
558
+192
558
558
382
382
+192
+192
+16
+16
+1
ΔRT, min
proposed reaction
−4.7
moxy + gluc
−4.4
moxy + gluc
−4.0
moxy + gluc
−3.0
moxy
−2.6
moxy
+2.2
hydrolysis
b
hepatocytes
human
rat
<1%
<1%
1.4%
2.5%
1.5%
1.0%
1.5%
<1%
29.1%
93.2%
66.8%
<1%
a
b
Percentage of metabolite present is after 4 h incubation with hepatocytes. Abbreviations: moxy, monooxygenation; gluc, glucuronidation.
Table 5. PDE4 Inhibitory Activity of 28 and 29 Compared to Apremilast (30)
PDE4D7-S129D
dimer activated
PDE4D7-S129(wt)
dimer basal
PDE4B1-S133D
dimer activated
PDE4D3-S54D
dimer activated
PDE4D2
monomer
PDE4D-cat
monomer
compd
parameter
IC50 (nM)
28
29
7.8 1.8
1018 239
0.58 0.11
2013 256
0.92 0.21
7.4 2.0
127 1.2
6561 10
Hill slope
0.55 0.12
0.68 0.14
0.60 0.16
1.2 0.10
c
a
c
c
b
c
c
I
max (%)
90.2 4.8
*
*
89 2.3
count
15
9
6
5
4
3
d
IC50 (nM)
Hill slope
0.8 1.6
0.66 0.03
88 3.6
2
885 2.2
128 1.3
−
−
−
−
−
−
−
−
−
−
−
−
d
0.50 0.1
0.37 0.04
c,
d
c
c
I
max (%)
d
count
2
2
30(apremilast) IC50 (nM)
26 1.3
1.0 0.14
97 3.5
8
32 1.6
1.2 0.3
96 2.4
6
24 1.2
1.0 0.05
23 1.2
1.1 0.14
98 2.0
5
35 1.3
1.1 0.09
99 1.0
4
21 1.2
1.1 0.11
98 1.8
3
Hill slope
I
max (%)
100
0
count
2
a
b
Two-Way ANOVA (F(1,29) = 27.3, p < 0.001; Sidak’s multiple comparisons test, p = 0.0012. Two-Way ANOVA (F(1,29) = 27.3, p < 0.001; Sidak’s
c
d
multiple comparisons test, p = 0.0019. *: Imax could not be calculated due to incomplete inhibition curve. −: not tested. Values tabulated are
mean SD. Count-the number of times that the compound was tested.
kg. Thus, the potential therapeutic window was estimated as 10-
fold. On the basis of these promising data, we focused chemical
optimization on improving solubility and metabolic stability.
To identify sites of metabolism, compound 23 was incubated
with rat or human hepatocytes and the products were
characterized (Table 4). Hepatocyte metabolism resulted in
the hydrolysis of the amide to a carboxylic acid and mono-
oxidation of the 6-ethyl side chain with further glucuronidation.
Hydrolysis to the acid was more extensive in rat (29.1%) than in
human hepatocytes (<1%), while both species produced mono-
oxygenated and glucuronidated products. We replaced the
amide with the acid to yield 28 and introduced a 6-CF3 on the
pyridine core to eliminate the possibility of side chain oxidation.
This also might balance chemical properties and could improve
solubility. As an alternative strategy to reduce hydrolysis of the
amide, we replaced the amide with an ethanolamide as in 29 with
the intention that the N-(2-hydroxyethyl)acetamide group
would reduce hydrolysis of the amide while also improving
solubility. Surprisingly, both compounds were potent and
selective PDE4D allosteric inhibitors (Table 5). Our previous
SAR had indicated that Ar1 carboxylic acid analogues were
neither potent nor selective for PDE4D in the triazine or
cyclopentylpyridine series (Table 1 and Table 2).
G
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Journal of Medicinal Chemistry
Drug Annotation
Figure 7. Comparison and selectivity of 23 and 28. (A) 23 with AR1 amide pointing up in the pocket to form water-mediated hydrogen bonds with the
main chain and side chain of UCR2 (cyan) threonine275 (T275) and catalytic domain glutamine451 (Q451), histidine506 (H506), and waters
surrounding the magnesium atom (green sphere). (B) 28 (PDB 6NJJ) with AR1 carboxylic acid pointing down losing water-mediated hydrogen bonds
with histidine506 (H506) and the side chain of threonine275 (T275) but gaining water-mediated hydrogen bonds to serine449 (S449), asparagine450
(N450), and the main-chain of glutamine451 (Q451), while maintaining the main chain interaction with UCR2 threonine275 (T275). (C) Modeling
of tyrosine271 (Y271) in place of phenylalanine271 (Y271) onto the crystal structure of PDE4D and 28. The closest approach by phenylalanine271 to
28 is 4.4 Å, which is reduced with tyrosine to 3.2 Å.
Figure 8. Kinetics of PDE4 enzyme inhibition contrasting a PDE4D subtype selective allosteric inhibitor (28, top row) with apremilast (30), an active
site-directed PDE4 inhibitor competitive with cAMP (bottom row). The low Hill slope of the allosteric inhibitor against the activated, dimeric forms of
PDE4D indicates negative cooperativity between the two active sites in the PDE4 dimer. Negative cooperativity is reduced (Hill slope tends toward 1)
when UCR2 is deleted as in the truncated PDE4D-cat enzyme. Apremilast inhibits equally well all PDE4 subtypes independent of their activation state
and does not require UCR2 for binding. Compounds were tested in duplicate wells using a coupled enzyme assay. The Z′ quality factor for the in vitro
inhibition assay was >0.6. Representative assays are shown plotting the well mean SD (note: the variance between duplicate wells may be less than
the size of the symbols).
The crystal structure of 28 shows binding of its core and Ar2
similar to 23, with differences in how the AR1 group engages the
UCR2 and catalytic domain (Figure 7). 28 was stable in human,
rat, mouse, and dog liver microsomes (88−100% remaining after
30 min incubation) with improved kinetic solubility (95 μM at
pH 7.4). Protein binding was 99.5−99.8% in plasma from
humans, rats, mice, and dogs, although there was no shift in
PDE4D inhibitory potency in the presence of BSA at 1 mg/mL.
28 had low potential for cytochrome P450 inhibition (CYP3A4
IC50 = 15 μM, 2C19 = 43 μM; 2D6 > 100 μM; 2C9 > 51 μM).
MDCK-MDR1 cellular permeability was high with low efflux
(A−B = 53 × 10−6 cm/s; B−A = × 10−6 cm/s; efflux ratio = 1.3)
despite the presence of the carboxylic acid. 28 lacked off target
activity against PDE1−11 (BPS Bioscience), a panel of G-
protein coupled receptors, ion channels, and transporters
(Cerep), or the human ERG cardiac channel with IC50 in all
assays >10 μM. The compound was not mutagenic in bacteria.
Evaluation of 28 in C57Bl6 mice showed high bioavailability (F
% = 82%) and prolonged half-life (10−12 h), although brain
distribution was moderate (brain/plasma ratio = 0.45). 28
provided cognitive benefit in the mouse NOR at doses above 0.3
mg/kg PO (plasma Cmax = 153 29 ng/mL, mean SD), while
no change in anesthesia duration was observed at doses up to 30
mg/kg (plasma Cmax = 11,475 3,570 ng/mL) (Figure 6).
Compound 29 showed reduced metabolic stability in liver
microsomes in contrast to 23 (e.g., 36% remaining in mouse
liver microsomes after 30 min incubation) with low kinetic
solubility at pH 7.4 (29 μM), had increased plasma protein
binding (99.6−99.8%), and was a weak CYP3A4 inhibitor (IC50
= 3.1 μM). 29 improved novel object discrimination in mice at a
dose of 3 mg/kg but also reduced the duration of anesthesia at
the same dose (Figure 6). Therefore, the compound was
abandoned.
We compared and contrasted the kinetics of PDE4 enzyme
inhibition for 28 as compared to apremilast (30) across a panel
of PDE4 enzymes (Figure 8). 28 was equipotent against two
dimeric forms of PDE4D containing activating mutations that
mimic UCR1 phosphorylation by PKA, PDE4D7-S129D, and
PDE4D5-S54D (Table 5). The two isoforms differ in the length
of the N-terminal sequence preceding UCR1 but are identical in
H
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Journal of Medicinal Chemistry
Drug Annotation
a
Scheme 1. Synthetic Schemes
a
Reagents and conditions: (a) EtCOCH2CO2Et, NaOMe; (b) POCl3; (c) H2NAr1, heat; (d) BrCH2Ar1, Pd(dppf)Cl2; (e) HOAr1, K2CO3; (f) 3-
Cl PhB(OH)2, Pd(Ph3P)4, K3PO4; (g) (1) HSAr1, Et3N, (2) m-CPBA;
sequence thereafter. 28 is 14-fold less potent against a
monomeric form of PDE4D, PDE4D2, which contains a
truncated UCR2 that retains the portion of UCR2 needed for
allosteric inhibitor binding. 28 is 137-fold selective for the
activated PDE4D7 enzyme as compared to the basal enzyme,
PDE4D7-S129(wt). Potency and selectivity against PDE4B1−
S133D is roughly similar (225-fold), while deletion of UCR2 as
in the PDE4D catalytic domain construct (PDE4D-cat) greatly
reduces inhibitory potency. For comparison, apremilast (30) has
no selectivity for PDE4 subtypes, inhibits equally dimeric and
monomeric isoforms of PDE4D, and does not require the
presence of UCR2. The steric bulk of the phthalate moiety in
apremilast projects toward solvent when the compound is
bound in the PDE4 active site and likely prevents closure of
UCR2 (Supporting Information, Figure 2).
We showed previously that PDE4D allosteric inhibitors
behave kinetically as partial inhibitors with Imax ∼ 90%.7 This
kinetic behavior is seen with 28 (Figure 8, Table 5). Imax is ∼90%
against the two dimeric, activated forms of PDE4D tested as
compared to apremilast (30) for which Imax = 100%. This
difference in kinetic behavior between 28 and 30 is statistically
significant (Two-Way ANOVA, F(1,29) = 27.3, p < 0.001). We
showed previously that the two active sites of the PDE4D dimer
exhibit negative cooperativity such that binding of inhibitor to
the first site reduces the affinity for the second site (Hill slope
<1).7 Binding of the allosteric inhibitor in the first site also
reduces the catalytic activity (Kcat) of the second site, so rather
than Imax = 50% (first site closed, second site open and fully
active), Imax = 90% due to the decrease in Kcat of the second site
(first site closed, second site open but only partially active, see
also Burgin et al. 20107). The kinetic behavior of 28 is altered
when UCR2 is removed as in the truncated PDE4D catalytic
domain (PDE4D-cat). Negative cooperativity is lost, 28 binding
is competitive with cAMP, and the Hill slope for the inhibition
curve tends toward to 1 (Figure 8).
Evaluation of 28 pharmacokinetics in rats and dogs showed
high oral bioavailability (F% 80−100%). Safety pharmacology
studies showed no adverse CNS or pulmonary effects in rats up
to a maximum oral dose of 60 mg/kg, and no effect on dog
cardiovascular function up to a maximum oral dose of 100 mg/
kg. Dose range finding toxicology studies demonstrated
inappetence and weight loss in rats at an oral dose of 100 mg/
kg and emesis, tremor, and weight loss in dogs at 100 mg/kg.
Toxicological studies of 28 and 90 days duration in rats and dogs
did not reveal vascular toxicity nor any other microscopic
findings in contrast to PDE4 inhibitors that lack subtype
selectivity. 28 did not cause emesis in ferrets at oral doses up to
30 mg/kg, nor did it cause emesis in cynomolgus monkeys at
oral doses up to 50 mg/kg. On the basis of this profile, 28 was
advanced into human clinical trials.
Compounds were synthesized as depicted in Scheme 1. 1,3,5-
Triazines 4−11 were assembled as previously described.49
Pyrimidines 22−27 and 29 were assembled from the amidine 30
by cyclization with an appropriate β-keto ester and chlorination
with POCl3 to yield 31, then displacement of the pyrimidine
chloride to obtain the desired Ar1 products. Pyridines 12−21
and 28 were synthesized by a regioselective Suzuki reaction of
32 to yield 33. Regioselective substitution of the pyridine
chloride yielded the desired Ar1 products 12−21. Regioselective
Suzuki reaction of 34 gave 35, which was then subjected to a
second Suzuki reaction with a benzyl boronate which afforded
28.
DISCUSSION
■
We have shown that it is possible to discover potent, subtype
selective, allosteric inhibitors of PDE4D. These exploit a key
selectivity residue in the UCR2 regulatory domain, a phenyl-
alanine in PDE4D versus a tyrosine in PDE4B. This key
selectivity residue is biologically significant as it affects KM for
substrate. PDE4D is more active at lower cAMP concentration
with a KM of 1.5 μM as compared to 7.7 μM for PDE4B, while
I
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Journal of Medicinal Chemistry
Drug Annotation
reciprocal exchange of the phenylalanine and the tyrosine
between the two PDE4 subtypes reverses the difference in KM.7
Modeling studies suggest that the tyrosine hydroxyl in PDE4B
may form a hydrogen bond with the cAMP ribose and that this is
absent when a phenylalanine is present on UCR2 as in PDE4D.
On the basis of these data, we offer the working hypothesis that
the two active sites in the PDE4 dimer communicate by rotation
around the four-helix bundle such that UCR2 alternately opens
and closes in trans- across the opposite active site during the
catalytic cycle. Capture of cAMP by the closure of UCR2
facilitates hydrolysis of the cyclic phosphodiester bond and
subsequent release of AMP when UCR2 reopens.
PDE4 allosteric inhibitors capture the dimer in a non-
productive conformation in which one active site is closed by
UCR2 while the other is open.7 The Kcat of the open active site is
reduced as the corresponding UCR2 is prevented from closing
over the cAMP substrate. Compounds with high affinity for both
the catalytic domain and UCR2 are able to fully inhibit the
dimeric enzyme by binding simultaneously in both active sites.
Binding in the closed active site is due to completion of a
hydrophobic surface that allows capping by UCR2, while
binding in the open active site is competitive with substrate and
does not require UCR2. Reducing affinity for the catalytic
domain, as when the linker is a methylene rather than an amine,
yields compounds with improved potency and selectivity for
PDE4D as well as for the activated state of the enzyme. Allosteric
inhibitors bind strongly in the closed active site, but due to
reduced affinity for the open active site, behave as partial
inhibitors with Imax = 90%. In this model, the N-terminus of
UCR1 likely regulates rotation around the four-helix bundle. We
hypothesize that this holds the dimer in a nonproductive
conformation which thereby reduces the basal activity of the
enzyme. Correspondingly, phosphorylation by PKA releases
inhibition by UCR1, allows rotation around the four-helix
bundle, and permits full enzyme activity. On the basis of the
genetic analysis of ACRDYS2 mutations, our therapeutic
hypothesis is that inhibition of the activated, dimeric forms of
PDE4D will be associated with cognitive benefit as this will
augment cAMP signaling while maintaining the spatial and
temporal patterning of information at the synapse.
that expresses PDE4D.67 Knockdown of PDE4D in normal mice
improves maturation of dendritic spines on cortical pyramidal
neurons.70
In separate studies, we have shown that 28 modulates
signaling through cAMP leading to activation of PKA and
phosphorylation of the CREB transcription factor.54 A challenge
in exploring PDE4D pharmacology in mice is the absence of the
key phenylalanine residue in UCR2 that is exploited for PDE4D
subtype selectivity. Instead, this is a tyrosine as in PDE4A-C.7
The core amino acid sequence of PDE4D from UCR1 through
the end of the catalytic domain is absolutely conserved across
mammalian species except for the phenylalanine in UCR2 which
is unique to primate.7 That single amino acid difference may be
an important biological adaption in primates that allows the
enzyme to function at lower cAMP concentrations, an adaption
in primates that may enhance dendritic compartmentalization in
cortical neurons.71
To better understand PDE4D pharmacology in mice, we
humanized the mouse PDE4D gene by replacing the single
codon encoding the key UCR2 tyrosine with the corresponding
codon for phenylalanine.54 The single amino change in the
humanized mouse enzyme lowers 28 IC50 from 133 18 to 2.9
0.3 nM. Correspondingly, 28 shows increased potency in
humanized PDE4D mice as compared to wild-type mice. 28
increases brain cAMP, increases phosphorylation of CREB,
augments the late phase of hippocampal long-term potentiation
(LTP), improves short and long-term memory, and increases
production of brain-derived neurotrophic factor (BDNF) in
hippocampus.54
CONCLUSION
■
Modification of this fundamental pathway of cognition has been
the target of extensive research over multiple decades but has not
been evaluated clinically because earlier compounds lacked
PDE4D subtype selectivity or could not allow for dosing within
the therapeutic range. Evaluation of 28 (BPN14770) will
provide the first proof-of-concept study for this therapeutic
mechanism in humans and, if successful, will facilitate the
development of a novel molecule for the treatment of Fragile X
Syndrome and other brain disorders.
One of the earliest biochemical findings in patients with
Fragile X Syndrome (FXS) was decreased production of cAMP
at baseline and in response to signaling through adenylate
cyclase.57−59 FXS is an X chromosome-linked, genetic disorder
associated with cognitive impairment, reduced IQ, and autistic
behavior.60 Subsequent cloning of the FMR1 gene responsible
for FXS allowed the creation of genetically exact models of the
disorder in the Drosophila fruit fly and in mice.61−63 In both
species, deletion of the FMR1 gene causes a decrease in baseline
and stimulated levels of cAMP production.64−66 This causes
defects in associative learning and neurodevelopment in the fly
model and global changes in behavior in the mouse model. The
FMR1 knockout mice show anxiety-related behaviors (increased
open field activity), impaired social interaction with wild-type
mice, and impairment of natural behaviors such as nest building
and marble burying.62,67 In patients, the FMR1 gene deletion
prevents the maturation of excitatory synapses in cortex and
hippocampus.68,69 Excitatory synapses occur on dendritic
spines. These remain thin and elongate in patients with FXS
and that defect also is seen in the mouse FMR1 knockout model.
We show separately that 28 normalizes the behavior of adult
FMR1 knockout mice and promotes the maturation of dendritic
spines on layer 2/3 pyramidal cells, a type of neuron in cortex
EXPERIMENTAL SECTION
■
General Synthetic Procedures. Unless otherwise noted, all
purchased reagents were used as received without further purification.
1H NMR spectra were recorded on a Bruker Avance III 400 MHz or
1
Bruker Avance III 500 MHz spectrometer. H NMR spectra were
reported in parts per million (ppm) downfield from tetramethylsilane.
In reported spectral data, the format (δ) chemical shift (multiplicity, J
values in Hz, integration) was used with the following abbreviations: s =
singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, m =
multiplet. Mass spectroscopy data were obtained on a Micromass LCT.
Compound HPLC retention times using the following HPLC methods:
Method 1: Agilent 1100 series, X-Bridge C18 column, 5−100% ACN/
water (0.07% H3PO4) (3.5 min), 100% ACN (0.07% H3PO4) (0.25
min), 100% to 5% ACN/water (0.07% H3PO4) (1.25 min) and 5%
ACN/water (0.07% H3PO4) (0.25 min), flow rate 2 mL/min. Method
2: Varian Star,Gemini-NX C18 column, 55% ACN (0.025% TFA)/
water (3 min), 55−90% ACN (0.025% TFA)/water (18 min), 90%
ACN (0.025% TFA)/water (2 min), 90−2% ACN (0.025% TFA)/
water (2 min). Method 3: Varian Star, Gemini-NX C18column, 2−90%
ACN (0.025% TFA)/water (18 min), 90% ACN (0.025% TFA)/water
(2 min), 90−2% ACN (0.025% TFA)/water (2 min), flow rate 1.0 mL/
min. Method 4: Varian Star, Phenomenex Luna C18(2)column, 10−
95% ACN/water (0. 1% TFA) (10 min), 95% ACN/water (0.1% TFA)
(6 min), flow rate 1.0 mL/min. Method 5: Varian Star, Phenomenex
J
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Drug Annotation
Luna C18(2) column, 5−95% ACN/water (0. 1% TFA) (15 min), 95%
ACN/water (0.1% TFA) (5 min), flow rate 0.75 mL/min. Method 6:
Varian Star, Phenomenex Luna C18(2) column, 10−90% ACN/water
(0.025% TFA) (10 min), 90% ACN/water (0.025% TFA) (10 min),
flow rate 1.0 mL/min. Unless otherwise stated compounds are ≥95%
determined by HPLC or elemental analysis.
General Procedure for the Synthesis of Compoound 4−8. To
a suspension of 2-chloro-4-(3-chlorophenyl)-6-ethyl-1,3,5-triazine (1
equiv) and appropriate aniline (1.5−2.0 equiv) were suspended in
HOAc and heated to 100 °C until the reaction was complete by HPLC.
The cooled reaction mixture was poured into water and the precipitate
collected and washed with water to obtain the desired product.
3-(4-((4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-yl)amino)-
phenyl)propan-1-ol (4). 1H NMR (DMSO-d6, 500 MHz) δ 10.19 (s,
1H), 8.41−8.31 (m, 2H), 7.71−7.67 (m, 3H), 7.60 (t, J = 8.0 Hz, 1H),
7.24−7.16 (m, 2H), 4.45 (t, J = 5.5 Hz, 1H), 3.43 (q, J = 5.5 Hz, 2H),
2.77 (q, J = 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 2.51−2.49 (m, 2H),
1.75−1.69 (m, 2H), 1.32 (t, J = 7.5 Hz, 3H). APCI MS m/z 369.2 [M +
H]+. HPLC method 2: retention time 12.85 min.
2-(4-(4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-ylamino)-
phenyl)ethanol (5). 1H NMR (CDCl3, 360 MHz) δ 8.46 (s, 1H), 8.35
(d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.50 (m, 1H), 7.43 (dd, J =
7.6 and 7.8 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 3.89 (m, 2H), 2.89 (m,
4H), 1.40 (t, J = 7.5 Hz, 3H). TOF MS ES+ m/z 355.1 [M + H]+.
HPLC method 1: retention time 3.23 min.
2-(4-(4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-ylamino)-
phenyl)acetamide (6). 1H NMR (CDCl3, 360 MHz) δ 8.44 (s, 1H),
8.35 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.50 (m, 1H), 7.42
(dd, J = 7.7 and 8.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 5.40 (br s, 2H),
3.59 (s, 2H), 2.84 (q, J = 7.5 Hz, 2H), 1.39 (t, J = 7.5 Hz, 3H). TOF MS
ES+ m/z 368.1 [M + H]+. HPLC method 1: retention time 2.89 min.
(4-((4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-yl)amino)phenyl)-
methanol (7). 1H NMR (DMSO-d6, 500 MHz) δ 10.24 (s, 1H), 8.42−
8.31 (m, 2H), 7.75−7.68 (m, 3H), 7.61 (t, J = 7.5 Hz, 1H), 7.36−7.28
(m, 2H), 5.11 (t, J = 5.5 Hz, 1H), 4.48 (d, J = 5.5 Hz, 2H), 2.78 (q, J =
7.5 Hz, 2H), 1.32 (t, J = 7.5 Hz, 3H). APCI MS m/z 341.1 [M + H]+.
HPLC method 2: retention time 10.79 min.
4-(4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-ylamino)-
benzamide (8). 1H NMR (DMSO-d6, 360 MHz,) δ 10.53 (s, 1H), 8.35
(m, 2H), 7.89 (br s, 4H), 7.70 (d, J = 7.7 Hz, 2H), 7.61 (dd, J = 7.8 and
8.0 Hz, 1H), 7.26 (br s, 2H), 2.82 (q, J = 7.5 Hz, 2H), 1.32 (t, J = 7.5 Hz,
3H). TOF MS ES+ m/z 354.1 [M + H]+. HPLC method 1: retention
time 2.95 min.
General Procedure for the Synthesis of Compounds 9−11. A
suspension of 2-chloro-4-(3-chlorophenyl)-6-ethyl-1,3,5-triazine (1
equiv) and the appropriate aniline (2.0 equiv) in HOAc was heated
to 100 °C until the reaction was complete by HPLC. The cooled
reaction mixture was diluted with saturated NaHCO3 and EtOAc. The
organic phase was dried over Na2SO4, filtered, and concentrated. The
crude material was purified by chromatography to obtain desired
product.
2-(4-(4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-ylamino)-
phenyl)propanoic Acid (9). 1H NMR (DMSO-d6, 360 MHz,) δ 12.26
(s, 1H), 10.28 (s, 1H), 8.32 (m, 2H), 7.73 (d, J = 8.4 Hz, 2H), 7.68 (d, J
= 7.7 Hz, 1H), 7.60 (dd, J = 7.7 and 7.9 Hz, 1H), 7.27 (d, J = 8.4 Hz,
2H), 3.64 (q, J = 7.1 Hz, 1H), 2.76 (q, J = 7.5 Hz, 2H), 1.35 (d, J = 7.1
Hz, 3H), 1.30 (t, J = 7.5 Hz, 3H). TOF MS ES+ m/z 353.3 [M + H]+.
HPLC method 1: retention time 3.34 min.
4-((4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-yl)amino)benzoic
Acid (10). 1H NMR (DMSO-d6, 360 MHz,) δ 10.65 (s, 1H), 8.36 (m,
2H), 7.95 (br s, 4H), 7.70 (d, J = 7.4 Hz, 1H), 7.61 (dd, J = 7.7 and 8.2
Hz, 1H), 2.81 (q, J = 7.5 Hz, 2H), 1.33 (t, J = 7.6 Hz, 3H). TOF MS ES+
m/z 355.1 [M + H]+. HPLC method 1: retention time 3.30 min.
2-(4-((4-(3-Chlorophenyl)-6-ethyl-1,3,5-triazin-2-yl)amino)-
phenyl)acetic Acid (11). 1H NMR (DMSO-d6, 360 MHz,) δ 10.29 (s,
1H), 8.32 (m, 2H), 7.58−7.73 (m, 4H), 7.24 (d, J = 7.4 Hz, 2H), 3.53
(s, 2H), 2.76−2.80 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.5 Hz, 3H). TOF
MS ES+ m/z 369.1 [M + H]+. HPLC method 1: retention time 3.19
min.
charged with 33 (30 mg, 0.1 mmol) and methyl 2-(4-aminophenyl)-
acetate (33 mg, 0.2 mmol) and the resulting mixture heated at 150 °C
under Ar for 1 h. After cooling to rt, the mixture was partitioned
between NaHCO3 (10 mL) and DCM (10 mL). The organic phase was
concentrated and the crude material purified by chromatography
(DCM/MeOH, 0−3%) to give methyl 2-[4-[[2-(3-chlorophenyl)-6,7-
dihydro-5H-cyclopenta[b]pyridin-4-yl]amino]phenyl]acetate (31 mg,
77% yield).
LAH (20 mg, 0.53 mmol, 10 equiv) was added portionwise to a 0 °C
solution of methyl 2-[4-[[2-(3-chlorophenyl)-6,7-dihydro-5H-
cyclopenta[b]pyridin-4-yl]amino]phenyl]acetate (20.7 mg, 0.053
mmol) in THF (2 mL). When the addition was complete, the reaction
was stirred under Ar at rt overnight. The reaction was quenched with 2
N HCl (aq) (2 mL) and saturated NaHCO3. The mixture was extracted
with EtOAc (3 × 5 mL) and the organic layers combined and
concentrated. The crude material was purified by chromatography
using (DCM/EtOAc, 0−30%) to give compound 12 (3.5 mg, 18%
yield). 1H NMR (CDCl3, 360 MHz) δ 7.84 (m, 2H), 7.41 (m, 3H), 7.21
(m, 3H), 7.16 (s, 1H), 5.95 (br s, 1H), 3.90 (m, 2H), 3.18 (m, 2H), 2.87
(m, 4H), 2.24 (m, 2H). TOF MS ES+ m/z 331.1 [M + H]+. HPLC
method 1: retention time 1.86 min.
3-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl)amino)phenyl)propanamide Hydrochloride (13).
A mixture of 33 and 3-(4-aminophenyl)propanamide (0.066 g, 0.40
mmol) in NMP (3 mL) was heated under microwave conditions at 120
°C for 3 h, then at 140 °C for 2.5 h. The reaction was diluted with
EtOAc, washed with water and brine, then dried with Na2SO4 and
concentrated. The crude material was purified by silica gel
chromatography (hexanes/EtOAc, 0−100%, then DCM/MeOH, 0−
5%). The resulting solids were further purified by preparative HPLC to
afford compound 13 (0.042 g, 54%) as a white solid. 1H NMR (DMSO-
d6, 500 MHz) δ 14.09 (br s, 1H), 9.82 (s, 1H), 7.90−7.89 (m, 1H),
7.71−7.58 (m, 3H), 7.33−7.27 (m, 5H), 6.98 (s, 1H), 6.77 (br s, 1H),
3.18−3.15 (m, 2H), 2.93−2.80 (m, 4H), 2.40−2.34 (m, 2H), 2.27−
2.21 (m, 2H). ESI MS m/z 392.0 [M + H]+. HPLC method 1: retention
time 1.81 min.
General Procedure for the Synthesis of Compoound 14 and
15. The appropriate aniline (3 equiv) and 33(1 equiv) were heated for
3 h at 150 °C. Toluene was added to the reaction, which was then
heated for an additional 7 h. After this time, the mixture was purified by
silica gel chromatography eluting with DCM and MeOH and the
resulting solid converted to the HCl salt.
2-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-
4-yl)amino)phenyl)-N,N-dimethylacetamide Hydrochloride (14). 1H
NMR (DMSO-d6, 500 MHz) δ 14.2 (br s, 1H), 9.88 (s, 1H), 7.91−7.90
(m, 1H), 7.72−7.66 (m, 2H), 7.61−7.58 (m, 1H), 7.37−7.32 (m, 1H),
7.01 (s, 1H), 3.73 (s, 2H), 3.17 (t, J = 7.7 Hz, 2H), 3.04 (s, 3H), 2.93 (t,
J = 7.2 Hz, 2H), 2.84 (s, 3H), 2.27−2.21 (m, 2H). ESI+ MS m/z 406.0
[M + H]+. HPLC method 3: retention time 11.83 min.
2-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-
4-yl)amino)phenyl)-N-methylacetamide Hydrochloride (15). 1H
NMR (DMSO-d6, 500 MHz) δ 14.07 (br s, 1H), 9.82 (s, 1H), 8.04−
8.03 (m, 1H), 7.90−7.89 (m, 1H), 7.71−7.58 (m, 3H), 7.38−7.34 (m,
4H), 7.01 (s, 1H), 3.44 (s, 2H), 3.18−3.14 (m, 2H), 2.93 (t, J = 7.3 Hz,
2H), 2.59 (d, J = 4.6 Hz, 3H), 2.27−2.21 (m, 2H); ESI MS m/z 392.0
[M + H]+.HPLC Method 3: retention time: 11.29 min.
2-(3-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl)amino)phenyl)ethanol Hydrochloride (16). Methyl
2-(3-aminophenyl)acetate (0.130 g, 0.79 mmol) and 33 (0.160 g, 0.53
mmol) in dioxane were treated with Pd(OAc)2 (5.8 mg, 0.026 mmol),
rac-BINAP (24 mg, 0.040 mmol), and Cs2CO3(430 mg, 1.33 mmol)
and, under Ar, was heated until 33 was consumed. The reaction was
diluted with water and extracted with EtOAc. The organic layer was
dried over Na2SO4, filtered, and concentrated. The residue was purified
by chromatography (hexanes/EtOAc, 0−100%) to afford the product
which then converted to the hydrochloride to afford methyl 2-(3-((2-
(3-chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)amino)-
phenyl)acetate hydrochloride (0.190 g, 91%) as a white solid. 1H NMR
(DMSO-d6, 500 MHz) δ 14.04 (s, 1H), 9.81 (s, 1H), 7.92 (t, J = 1.8 Hz,
1H), 7.76−7.71 (m, 1H), 7.69−7.64 (m, 1H), 7.60 (t, J = 7.9 Hz, 1H),
2-[4-[[2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl]amino]phenyl]ethanol (12). An 18 mL test tube was
K
DOI: 10.1021/acs.jmedchem.9b00193
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
7.44 (t, J = 7.9 Hz, 1H), 7.38−7.34 (m, 1H), 7.33−7.28 (m, 1H), 7.23−
7.17 (m, 1H), 7.09 (s, 1H), 3.77 (s, 2H), 3.62 (s, 3H), 3.16 (t, J = 7.5
Hz, 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.24 (quin, J = 7.5 Hz, 2H). APCI MS
m/z 393 [M + H]+.
DCM followed by EtOAc to give 2-[4-[[2-(3-chlorophenyl)-6,7-
dihydro-5H-cyclopenta[b]pyridin-4-yl]amino]phenyl]acetonitrile (16
mg, 44% yield). 1H NMR (CDCl3, 360 MHz) δ 7.84 (s, 1H), 7.68 (m,
1H), 7.32 (m, 4H), 7.21 (m, 2H), 7.15 (s, 1H), 5.82 (br s, 1H), 3.75 (s,
2H), 3.09 (t, J = 7.50 Hz, 2H), 2.83 (t, J = 7.20 Hz, 2H), 2.21 (m, 2H).
Con H2SO4 (0.5 mL) was added at 0 °C to an 18 mL vial containing
2-[4-[[2-(3-chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-
yl]amino]phenyl]acetonitrile (12 mg, 0.033 mmol) and stirred at rt for
6 h.. The reaction was slowly poured onto 0 °C Na2CO3. The resulting
precipitate was collected and washed with water, then dried. The solid
was treated with 4 N HCl in dioxane and the volatile material was
removed to give compound 19. (2.6 mg, 19% yield). 1H NMR (CDCl3,
360 MHz) δ 7.83 (s, 1H), 7.69 (m, 1H), 7.18−7.30 (m, 7H), 5.77 (br s,
1H), 5.44 (br s, 2H), 3.58 (s, 2H), 3.08 (t, J = 7.70 Hz, 2H), 2.83 (t, J =
7.10 Hz, 2H), 2.21 (m, 2H). ESI+ MS m/z 378.2 [M + H]+. HPLC
method 3: retention time 11.37 min.
2-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl)sulfonyl)phenyl)acetamide (20). A mixture of 33
(0.120 g, 0.40 mmol), methyl 2-(4-mercaptophenyl)acetate (0.100 g,
0.55 mmol), and Et3N (0.145 g, 1.3 mmol) in DMF was heated at 110
°C for 24 h. The reaction was concentrated, diluted with EtOAc (100
mL), washed with 10% aqueous LiCl (2 × 100 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by chromatography
(hexane/EtOAc, 0 to 50%) to afford methyl 2-(4-((2-(3-chlorophen-
yl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)thio)phenyl)acetate
(0.164 g, 100%) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 7.78−
7.77 (m, 1H), 7.61−7.46 (m, 3H), 7.37 (d, J = 8.2 Hz, 2H), 7.30−7.28
(m, 3H), 6.89 (s, 1H), 3.73 (s, 3H), 3.70 (s, 2H), 3.09 (t, J = 7.7 Hz,
2H), 2.90 (t, J = 7.4 Hz, 2H), 2.24−2.05 (m, 4H). APCI MS m/z 410
[M + H]+.
To a solution of methyl 2-(3-((2-(3-chlorophenyl)-6,7-dihydro-5H-
cyclopenta[b]pyridin-4-yl)amino)phenyl)acetate (0.080 g, 0.20 mmol)
in THF at 0 °C was added, BH3·DMS (2.0 M, 0.30 mL, 0.60 mmol).
The reaction was warmed to rt and stirred until starting material was
consumed by HPLC. The reaction was quenched with 0.5 M HCl,
diluted with saturated NaHCO3, and extracted with EtOAc. The
organic layer was dried over Na2SO4, filtered, and concentrated. The
crude material was purified by chromatography (hexanes/EtOAc, 0−
100%) and the isolated material treated with HCl in MeOH to give
compound 16 (0.064 g, 80%) as a light-yellow solid. 1H NMR (DMSO-
d6, 500 MHz) δ 14.08(s, 1H), 9.81 (s, 1H), 7.90 (t, J = 1.8 Hz, 1H),
7.76−7.68 (m, 1H), 7.67−7.63 (m, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.40
(t, J = 7.8 Hz, 1H), 7.30−7.27 (m, 1H), 7.26−7.23 (m, 1H), 7.20−7.16
(m, 1H), 7.09 (s, 1H), 3.65 (t, J = 6.5 Hz, 2H), 3.16 (t, J = 7.5 Hz, 2H),
2.92 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.24 (quin, J = 7.5 Hz,
2H). APCI MS m/z 365.2 [M + H]+. HPLC Method 3: retention time
11.92 min.
2-[4-[[2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl]amino]phenyl]acetic Acid (17). Methyl 2-[4-[[2-(3-
chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl]amino]-
phenyl]acetate (31 mg, 0.77 mmol) and LiOH (48 mg, 1.14 mmol) in
THF/water (2:1, 1.5 mL) were stirred at rt overnight. Then 2 N HCl
(0.6 mL) was added and the volatiles removed. The crude product was
purified by chromatography (DCM/MeOH, 2−5%) to give compound
1
17 (26 mg, HCl salt, 81% yield). H NMR (DMSO-d6, 360 MHz) δ
14.23 (br s, 1H), 12.39 (br s, 1H), 9.84 (br s, 1H), 7.91 (s, 1H), 7.70 (d,
J = 7.70 Hz, 1H), 7.63 (d, J = 8.40, 1H), 7.57 (dd, J = 7.70, 8.40 Hz, 1H),
7.36 (br s, 4H), 7.02 (s, 1H), 3.60 (s, 2H), 3.11 (t, J = 7.90 Hz, 2H),
2.90 (t, J = 7.40 Hz, 2H), 2.20 (m, 2H). TOF MS ES+ m/z 378.9 [M +
H]+. HPLC method 1: retention time 2.01 min.
A solution of methyl 2-(4-((2-(3-chlorophenyl)-6,7-dihydro-5H-
cyclopenta[b]pyridin-4-yl)thio)phenyl)acetate (0.164 g, 0.40 mmol)
and m-CPBA (0.269 g, 1.2 mmol) in DCM (15 mL) was stirred at rt for
2 h. The reaction was diluted with DCM (100 mL), washed with water
(100 mL) and saturated NaHCO3 (100 mL), dried over MgSO4,
filtered, and concentrated. The crude material was purified by
chromatography (hexane/EtOAc, 0−50%) to afford methyl 2-(4-((2-
(3-chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)-
2-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl)methyl)phenyl)acetamide Hydrochloride (18). A
10 mL sealed tube was charged with 33 (0.100 g, 0.33 mol), T-1 (0.096
g, 0.33 mmol), Pd(dppf)Cl2 (0.027 g, 0.033 mmol), and powdered
Na2CO3 (0.141 g, 1.33 mmol). Dioxane (3 mL) and water (1.5 mL)
were added and the mixture stirred under Ar at 90 °C for 3 days until 33
was consumed. The reaction was cooled and purified by chromatog-
raphy (hexanes/EtOAc, 0−100%) to afford methyl 2-(4-((2-(3-
chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)methyl)-
phenyl)acetate (0.036 g, 48%) as an off-white solid. 1H NMR (CDCl3,
500 MHz) δ 7.94−7.91 (m, 1H), 7.77 (dt, J = 7.0, 2.0 Hz, 1H), 7.36−
7.30 (m, 2H), 7.24 (s, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz,
2H), 3.95 (s, 2H), 3.69 (s, 3H), 3.60 (s, 2H), 3.08 (t, J = 8.0 Hz, 2H),
2.86 (t, J = 7.5 Hz, 2H), 2.18−2.09 (m, 2H). APCI MS m/z 392 [M +
H]+.
A 10 mL vial was charged with methyl 2-(4-((2-(3-chlorophenyl)-
6,7-dihydro-5H-cyclopenta[b]pyridin-4-yl)methyl)phenyl)acetate
(0.036 g, 0.09 mmol) and NH4Cl (0.015 g, 0.27 mmol). To this was
added MeOH (2 mL) followed by 7N NH3/MeOH (4 mL, 27.6
mmol). The vial was sealed and heated at 100 °C for 48 h. The reaction
was concentrated and purified by chromatography (hexanes/EtOAc
(0−100%). The isolated material was treated with HCl in MeOH to
afford the compound 18 (0.037 g, 99%) as a white solid; mp 178−180
°C. 1H NMR (DMSO-d6, 500 MHz) δ 8.11−8.07 (m, 1H), 8.00−7.93
(m, 1H), 7.81 (s, 1H), 7.57−7.47 (m, 2H), 7.43 (br s, 1H), 7.25−7.34
(m, 4H), 6.84 (br s, 1H), 4.60 (br s, 1H), 4.00 (s, 2H), 3.31 (s, 2H),
3.00 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.07 (q, J = 7.5 Hz,
2H). APCI MS m/z 377.4 [M + H]+. HPLC method 3: retention time
12.20 min.
1
sulfonyl)phenyl)acetate (0.109 g, 62%) as a white solid. H NMR
(CDCl3, 500 MHz) δ 8.09−8.0 (m, 2H), 7.97−7.85 (m, 3H), 7.57−
7.39 (m, 4H), 3.75−3.68 (m, 5H), 3.18−3.07 (m, 4H), 2.22−2.05 (m,
2H). APCI MS m/z 442 [M + H]+.
A suspension of methyl 2-(4-((2-(3-chlorophenyl)-6,7-dihydro-5H-
cyclopenta[b]pyridin-4-yl)sulfonyl)phenyl)acetate (0.109 g, 0.25
mmol) in 7N NH3/MeOH was heated at 100 °C in a sealed tube.
After 24 h, the mixture was concentrated and purified by
chromatography (DCM/(89:9:1, DCM/MeOH/con NH4OH), 0−
50%) to afford compound 20 (0.055 g, 53%) as a white solid. 1H NMR
(DMSO-d6, 500 MHz) δ 8.20−8.18 (m, 2H), 8.10−8.08 (m, 1H), 8.03
(d, J = 8.4 Hz, 2H), 7.56−7.51 (m, 5H), 6.98 (s, 1H), 3.50 (s, 2H), 3.12
(t, J = 7.6 Hz, 2H), 3.03 (t, J = 7.8 Hz, 2H), 2.13−2.08 (m, 2H). APCI
MS m/z 427.3 [M + H]+. HPLC method 3: retention time 18.03 min.
2-(4-((2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl)(methyl)amino)phenyl)acetamide (21). A mixture
of 33 (0.060 g, 0.20 mmol) and 2-(4-(methylamino)phenyl)acetamide
(0.066 g, 0.40 mmol) in NMP (3 mL) was microwaved for 3 h at 140
°C. The reaction was diluted with EtOAc, washed with water and brine,
then dried with Na2SO4 and concentrated. The crude material was
purified by silica gel chromatography (hexanes/EtOAc, 0−100%, then
DCM/MeOH, 0−5%). The resulting solids were further purified by
preparative HPLC to afford the title compound 21 (0.012 g, 15%) as a
white solid. 1H NMR (DMSO-d6, 500 MHz) δ 14.18 (br s, 1H), 8.10 (s,
1H), 7.94 (d, J = 7.5 Hz, 1H), 7.70−7.66 (m, 2H), 7.55 (br s, 1H),
7.37−7.27 (m, 5H), 6.92 (br s, 1H), 3.57 (s, 3H), 3.01 (t, J = 7.1 Hz,
2H), 1.96−1.85 (m, 4H). ESI MS m/z 392.1 [M + H]+. HPLC method
4: retention time 8.80 min.
2-[4-[[2-(3-Chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]-
pyridin-4-yl]amino]phenyl]acetamide Hydrochloride (19). An
18 mL test tube was charged with 33 (30 mg, 0.1 mmol) and 2-(4-
aminophenyl)acetonitrile (30 mg, 0.23 mmol), then heated at 150 °C
under Ar for 1 h. After cooling to rt, the reaction was diluted with
NaHCO3 (10 mL) and DCM (10 mL). The organic layer was
concentrated and the crude material purified by chromatography using
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)amino)-
phenyl)ethanol (22). A mixture of 31 (0.150 g, 0.59 mmol), 2-(4-
aminophenyl)ethanol (0.134 g, 0.89 mmol), and 4 M HCl in dioxane
(0.222 mL, 0.89 mmol) in EtOH was heated for 2.5 h at 85 °C. The
L
DOI: 10.1021/acs.jmedchem.9b00193
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
mixture was poured onto NaHCO3 at 0 °C and extracted with EtOAc.
The combined organic layers were dried with Na2SO4 and
concentrated. The crude residue was purified by silica gel
chromatography (DCM/MeOH, 0−5%) to afford compound
compoound 25 (17 mg, 36% yield) as a colorless oil. 1H NMR
(DMSO-d6, 500 MHz) δ 8.38−8.33 (m, 2H), 7.61−7.53 (m, 2H),
7.28−7.25 (m, 3H), 7.16 (d, J = 8.0 Hz, 2H), 4.58 (t, J = 5.5 Hz, 1H),
4.08 (s, 2H), 3.60−3.53 (m, 2H), 2.77 (q, J = 7.5 Hz, 2H), 2.67 (t, J =
7.0 Hz, 2H), 1.26 (t, J = 7.5 Hz, 3H). ESI MS m/z 353.3 [M + H]+.
HPLC method 3: retention time 21.65 min.
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)amino)-
phenyl)acetamide (26). A mixture of 31 (0.150 g, 0.59 mmol) and 2-
(4-aminophenyl)acetamide (0.107 g, 0.71 mmol) and 4 M HCl in
dioxane (2 drops) in HOAc (3 mL) was heated for 2 h at 108 °C. The
mixture was cooled to rt, neutralized with saturated NaHCO3, extracted
with EtOAc, dried over Na2SO4, filtered, and concentrated. The residue
was purified by chromatography (DCM/MeOH, 0−5%) to afford the
title compound 26 (0.135 g, 62%) as a white solid. 1H NMR (DMSO-
d6, 500 MHz) δ 9.57 (s, 1H), 8.33−8.29 (m, 2H), 7.64 (d, J = 8.4 Hz,
2H), 7.58−7.54 (m, 2H), 7.43 (br s, 1H), 7.27 (d, J = 8.5 Hz, 2H), 6.86
(br s, 1H), 6.59 (s, 1H), 3.35 (s, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.26 (t, J
= 7.6 Hz, 3H). ESI MS m/z 367.0 [M + H]+. HPLC method 5:
retention time 10.14 min.
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)oxy)-
phenyl)acetamide (27). A solution of 31 (0.200 g, 0.79 mmol),
methyl 2-(4-hydroxyphenyl)acetate (0.131 g, 0.79 mmol), and K2CO3,
(0.546 g, 3.95 mmol) in ACN (5 mL) was heated to 85 °C for 7 h. The
reaction mixture was diluted with water and extracted with EtOAc. The
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The residue was purified by chromatography (hexanes/
DCM, 0−100%) to afford methyl 2-(4-((2-(3-chlorophenyl)-6-ethyl-
pyrimidin-4-yl)oxy)phenyl)acetate (0.130 g, 65%). 1H NMR (CD3OD,
500 MHz) δ 8.21 (t, J = 1.8 Hz, 1H), 8.16−8.12 (m, 1H), 7.46−7.35
(m, 4H), 7.21−7.17 (m, 2H), 6.77(s, 1H), 3.73 (s, 2H), 3.72 (s, 3H),
2.83 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 6.4 Hz, 3H). APCI MS m/z 383 [M
+ H]+.
Methyl 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-4-yl)oxy)-
phenyl)acetate (0.075 g, 0.20 mmol) and 7 M NH3/MeOH (3 mL)
were heated at 100 °C for 72 h. The reaction was cooled, evaporated,
and purified by column chromatography (DCM/MeOH, 0−5%) to
afford compound 27 (0.048 g, 64%) as a white solid. 1H NMR (DMSO-
d6, 500 MHz) δ 8.16−8.13 (m, 1H), 8.14−8.11 (m, 1H), 7.59−7.55
(m, 1H), 7.51 (t, J = 7.9 Hz, 2H), 7.39−7.36 (m, 2H), 7.24−7.21 (m,
2H), 6.92 (s, 1H), 6.90 (s,1H), 3.44 (s, 2H), 2.80 (q, J = 7.6 Hz, 2H),
1.28 (t, J = 7.6 Hz, 3H). APCI MS m/z 368 [M + H]+. HPLC method 4:
retention time 13.32 min.
P r e p a r a t i o n o f 2 - ( 4 - ( ( 2 - ( 3 - C h l o r o p h e n y l ) - 6 -
(trifluoromethyl)pyridin-4-yl)methyl)phenyl)acetic Acid (28).
In a 250 mL round bottomed flask, 35 (1.66 g, 5.67 mmol), T-1 (1.89 g,
6.52 mmol), Na2CO3 (2.92 g, 27.5 mmol), and Pd(dppf)Cl2·DCM
(460 mg, 0.56 mmol) in dioxane (30 mL) and H2O (15 mL) were
purged with Ar for 20 min, then sealed and stirred at 90 °C for 4 h. The
reaction was concentrated to dryness and residue chromatographed
(hexanes/DCM, 2−50%) to afford methyl 2-(4-((2-(3-chlorophenyl)-
6-(trifluoromethyl)pyridin-4-yl)methyl)phenyl)acetate (1.76 g, 74%)
as a white solid; mp 72−74 °C. 1H NMR (CDCl3, 500 MHz) δ 8.03−
8.00 (m, 1H), 7.91−7.86 (m, 1H), 7.68 (s, 1H), 7.46−7.44 (m, 1H),
7.42−7.37 (m, 2H), 7.29−7.26 (m, 2H), 7.18−7.15 (m, 2H), 4.09 (s,
2H), 3.70 (s, 3H), 3.62 (s, 2H). APCI MS m/z 420 [M + H]+.
Methyl 2-(4-((2-(3-chlorophenyl)-6-(trifluoromethyl)pyridin-4-
yl)methyl)phenyl)acetate (1.75 g, 4.18 mmol) was dissolved in
MeOH/THF/H2O (1:1:1, 45 mL), treated with LiOH·H2O (1.0 g,
24 mmol), and stirred at rt overnight. The reaction was concentrated,
diluted with H2O (100 mL) and the pH adjusted to 3 with 2 N HCl.
The solid was collected and dried to yield 28 (1.58 g, 90%) as a white
solid; mp 124−126 °C. 1H NMR (CDCl3, 500 MHz) δ 8.03−8.00 (m,
1H), 7.91−7.85 (m, 1H), 7.68 (s, 1H), 7.46−7.44 (m, 1H), 7.42−7.37
(m, 2H), 7.30−7.26 (m, 2H), 7.19−7.15 (m, 2H), 4.09 (s, 2H), 3.65 (s,
2H). APCI MS m/z 406 [M + H]+. Elemental analysis for
C21H15ClF3NO2: Calculated C (62.16%), H (3.73%), Cl (8.74%), F
(14.05%), N (3.45%). Found: C (62.20%), H (3.55%), Cl (8.77%), F
(14.43%), N (3.32%).
1
22(0.145 g, 70%) as a yellow solid. H NMR (DMSO-d6, 500 MHz)
δ 9.53 (s, 1H), 8.33−8.28 (m, 2H), 7.62−7.55 (m, 4H), 7.22 (d, J = 8.4
Hz, 2H), 6.58 (s, 1H), 4.62 (t, J = 5.2 Hz, 1H), 3.63−3.59 (m, 2H),
2.72−2.64 (m, 4H), 1.26 (t, J = 7.6 Hz, 3H). ESI MS m/z 354.0 [M +
H]+. HPLC method 5: retention time 10.85 min.
Preparation of 2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-
4-yl)methyl)phenyl)acetamide (23). A mixture of 31 (870 mg, 3.44
mol), T-1 (1.00 g, 3.44 mmol), Pd(dppf)Cl2 (280 mg, 0.34 mmol), and
powdered Na2CO3 (1.09 g, 10.3 mmol) in dioxane (16 mL) and water
(8 mL) was heated at 90 °C under Ar for 2 h. The reaction was cooled to
rt, then filtered through Celite and the pad washed well with EtOAc.
The filtrate was washed with brine (3 × 25 mL), dried over Na2SO4,
filtered, and concentrated. The residue (1.8 g) was purified by
chromatography (hexanes/DCM, 0−100%, then hexanes/EtOAc (0−
24%) to afford methyl 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-4-
1
yl)methyl)phenyl)acetate (0.65 g, 50% yield) as a colorless oil. H
NMR (CDCl3, 500 MHz) δ 8.49−8.47 (m, 1H), 8.37 (dt, J = 7.5, 1.5
Hz, 1H), 7.44−7.38 (m, 2H), 7.31−7.27 (m, 2H), 7.27−7.23 (m, 2H,
overlaps with CDCl3), 6.85 (s, 1H), 4.10 (s, 2H), 3.69 (s, 3H), 3.61 (s,
2H), 2.76 (q, J = 7.5 Hz, 2H), 1.31 (t, J = 7.5 Hz, 3H).
In a 20 mL vial, methyl 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-
4-yl)methyl)phenyl)acetate (113 mg, 0.30 mmol) and NH4Cl (48 mg,
0.89 mmol) were suspended in MeOH (3 mL) and treated with 7N
NH3/MeOH (7.5 mL, 53 mmol). The vial was sealed and stirred at 100
°C for 43 h. The crude material was purified by chromatography
(DCM/MeOH, 0−5%; then hexanes/EtOAc, 0−100%) to give
compound 23 (37 mg, 35% yield) as a white solid. 1H NMR
(DMSO-d6, 500 MHz) δ 8.38−8.33 (m, 2H), 7.61−7.53 (m, 2H), 7.41
(br s, 1H), 7.29 (d, J = 8.0 Hz, 2H), 7.25 (s, 1H), 7.20 (d, J = 8.0 Hz,
2H), 6.82 (br s, 1H), 4.09 (s, 2H), 3.32 (s, 2H), 2.77 (q, J = 7.5 Hz, 2H),
1.26 (t, J = 7.5 Hz, 3H). APCI MS m/z 366.3 [M + H]+. HPLC method
3: retention time 19.54 min.
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)oxy)-
phenyl)ethanol (24). A solution of 31 (0.200 g, 0.79 mmol), methyl
2-(4-hydroxyphenyl)acetate (0.131 g, 0.79 mmol), and K2CO3 (0.546
g, 3.95 mmol) in ACN (5 mL) was heated to 85 °C for 7 h. The reaction
mixture was diluted with water and extracted with EtOAc. The organic
layer was dried over Na2SO4, filtered, and concentrated. The residue
was purified by chromatography (hexanes/DCM, 0−100%) to afford
methyl 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-4-yl)oxy)phenyl)-
acetate (0.130 g, 65%). 1H NMR (CD3OD, 500 MHz) δ 8.21 (t, J = 1.8
Hz, 1H), 8.16−8.12 (m, 1H), 7.46−7.35 (m, 4H), 7.21−7.17 (m, 2H),
6.77(s, 1H), 3.73 (s, 2H), 3.72 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 1.35 (t,
J = 6.4 Hz, 3H). APCI MS m/z 383 [M + H]+.
To a solution of methyl 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimi-
din-4-yl)oxy)phenyl)acetate (0.075 g, 0.20 mmol) in THF (1.3 mL) at
0 °C was added BH3·SMe2 (0.031 g, 0.39 mmol) and the mixture stirred
at rt overnight. The reaction was quenched with 0.5 M HCl, diluted
with NaHCO3, and extracted with EtOAc. The organic layer was dried
over Na2SO4, filtered, and concentrated. The residue was purified by
chromatography (hexanes/DCM, 0−100%) to afford compound 24 as
a white solid (0.052 g, 69%). 1H NMR (DMSO-d6, 500 MHz) δ 8.17−
8.10 (m, 2H), 7.59−7.54 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.37−7.32
(m, 2H), 7.22−7.17 (m, 2H), 6.88 (s, 1H), 4.67 (t, J = 5.2 Hz, 1H),
3.68−3.62 (m, 2H), 2.79 (q, J = 7.3 Hz, 4H), 1.28 (t, J = 7.6 Hz, 3H).
APCI MS m/z 355.1 [M + H]+. HPLC method 4: retention time 14.83
min.
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)methyl)-
phenyl)ethanol (25). A solution of 31 (52 mg, 0.13 mmol) and THF
(3 mL) at 0 °C was treated with a solution of 1 M LAH in THF (0.31
mL, 0.31 mmol) then slowly warmed to rt. After 4.5 h, the mixture was
quenched with MeOH, diluted with water (10 mL), and acidified with 2
N HCl to pH ∼ 3. It was extracted with EtOAc (3 × 12 mL) and the
combined organic layers washed with saturated brine (5 mL), dried
over Na2SO4, filtered, and concentrated. The crude material was
purified by chromatography (hexanes/EtOAc, 0−50%) to afford
2-(4-((2-(3-Chlorophenyl)-6-ethylpyrimidin-4-yl)methyl)-
phenyl)-N-(2-hydroxyethyl)acetamide (29). Methyl 2-(4-((2-(3-
M
DOI: 10.1021/acs.jmedchem.9b00193
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Journal of Medicinal Chemistry
Drug Annotation
chlorophenyl)-6-ethylpyrimidin-4-yl)methyl)phenyl)acetate (64 mg,
0.17 mmol, 1.0 equiv) and LiOH·H2O (21 mg, 0.50 mmol, 3.0 equiv)
were stirred in THF (3 mL) and water (3 mL) at rt until the starting
material was consumed. The reaction was diluted with water (10 mL)
and acidified with 2 N HCl. It was extracted with EtOAc, which was
washed with brine (5 mL), dried over Na2SO4, filtered, and
concentrated. The residue was purified by chromatography (DCM/
MeOH, 0−10%) to afford 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimi-
din-4-yl)methyl)phenyl)acetic acid (40 mg, 66% yield) as a white solid.
1H NMR (DMSO-d6, 500 MHz) δ 12.31 (br s, 1H), 8.40−8.33 (m,
2H), 7.62−7.53 (m, 2H), 7.31(d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 7.21 (d,
J = 8.0 Hz, 2H), 4.10 (s, 2H), 3.51 (s, 2H), 2.78 (q, J = 7.5 Hz, 2H), 1.26
(t, J = 7.5 Hz, 3H).
min. The reaction was sealed and stirred under Ar at 90 °C for 4 h. After
cooling to rt, the reaction mixture was diluted with EtOAc (100 mL)
and hexanes (100 mL). The aqueous layer was separated and the
organic layer washed with brine (2 × 25 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by chromatography
(hexanes) to afford 35 (1.82 g, 67%) as a white solid. 1H NMR (CDCl3,
500 MHz) δ 8.06−8.04 (m, 1H), 7.92 (dt, J = 7.0, 2.0 Hz, 1H), 7.89 (d,
J = 1.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.48−7.42 (m, 2H).
Methyl 2-(4-((4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
methyl)phenyl)acetate (T-1). Methyl 2-(4-(bromomethyl)phenyl)-
acetate (3.45 g, 14.2 mmol), pinacol diborane (4.32 g, 17.0 mmol),
Pd(PPh3)4 (1.64 g, 1.42 mmol), and K2CO3 (5.88 g, 42.6 mmol) in
dioxane (80 mL) were stirred under Ar at 80 °C for 23 h. The reaction
was cooled and diluted with EtOAc (200 mL) before it was filtered
through Celite. The filtrate was washed with brine (3 × 25 mL), dried
over Na2SO4, filtered, and concentrated. The crude material was
purified by chromatography (hexanes/EtOAc, 0−100%) to afford
methyl 2-(4-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)-
A solution of 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-4-yl)-
methyl)phenyl)acetic acid (0.22 g, 0.60 mmol) in DCM (15 mL) at
0 °C was treated with oxalyl chloride (0.25 mL, 3.00 mmol) followed by
DMF (1 drop). After 4 h, the volatile material was removed to afford
crude 2-(4-((2-(3-chlorophenyl)-6-ethylpyrimidin-4-yl)methyl)-
phenyl)acetyl chloride as a red oil. This was dissolved in DCM (8
mL), cooled to 0 °C, then treated with ethanolamine (0.060 mL, 1.00
mmol) and DIEA (0.17 mL, 1.00 mmol). After 3 h, the reaction mixture
was diluted with DCM (50 mL) and water (5 mL), then the pH was
adjusted to 5 with 2 N HCl. The organic layer was washed with brine (5
mL), dried over Na2SO4, filtered, and concentrated. The crude material
was purified by chromatography (hexanes/EtOAc, 0−100%) followed
by preparative HPLC (water/acetonitrile with 0.05% TFA) to afford
1
phenyl)acetate (2.88 g, 70% yield) as a colorless semisolid. H NMR
(CDCl3, 500 MHz) δ 7.14 (s, 4H), 3.67 (s, 3H), 3.57 (s, 2H), 2.27 (s,
2H), 1.23 (s, 12H).
Protein Expression and Purification. PDE4D residue numbering
was based on the reference PDE4D7 isoform, NCBI Reference
Sequence: NP_001159371.1. Human PDE4D7 contained a mutation
of S129D to mimic activation by cAMP-dependent protein kinase A
(PKA), PDE4D3 contained the corresponding mutation S54D, and
PDE4B1 contained the mutation S133D. All PDE4 proteins contained
the mutations S579A and S581A to remove the potential for ERK-
dependent phosphorylation. Methods used to generate synthetic genes
for human PDE4 subtypes and isoforms were as described previously.7
Synthetic genes were engineered with carboxyl- or amino-terminal
hexahistidine tags for baculovirus-infected insect cell expression and
purification.
1
compound 29 (0.036 g, 45%) as a white solid; mp 136−138 °C. H
NMR (DMSO-d6, 500 MHz) δ 8.37−8.31 (m, 2H), 8.02−7.96 (m,
1H), 7.59 (dt, J = 6.5, 2.0 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.28 (d, J =
8.0 Hz, 2H), 7.25 (s, 1H), 7.20 (d, J = 8.0 Hz, 2H), 4.63 (t, J = 5.0 Hz,
1H), 4.09 (s, 2H), 3.40−3.35 (m, 4H), 3.90 (q, J = 6.0 Hz, 2H), 2.77 (q,
J = 7.5 Hz, 2H), 1.26 (t, J = 7.5 Hz, 3H). APCI MS m/z 410.6 [M + H]+.
HPLC method 6: retention time 17.42 min.
PDE4 Enzyme Assay Protocol. Kinetic assay of cAMP hydrolysis
by purified PDE4 was measured by coupling the formation of the PDE4
reaction product, 5′-adenosine monophosphate to the oxidation of
reduced nicotinamide adenine dinucleotide (NADH) by the use of
three coupling enzymes (yeast myokinase, pyruvate kinase, and lactate
dehydrogenase), which allowed fluorescent determination of reaction
rates.7 Assays were performed in 96-well plates in a total volume of 200
μL/well. Compounds were dissolved in dimethyl sulfoxide (DMSO)
and added to plates in a volume of 10 μL followed by addition of 165 μL
of assay mix. Plates were preincubated at 25 °C for 5−10 min, and the
reactions were initiated by the addition of 25 μL of cAMP followed by
thorough mixing. Reaction rates were measured by monitoring the
decrease in fluorescence using excitation at 355 nm and emission at 460
nm for a period of 10 min in a fluorescence plate reader. Initial rates
(slopes) were determined from linear portions of the progress curves.
Final concentrations of assay components were as follows: 50 mM Tris,
pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-
carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP),
0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM
cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units
lactate dehydrogenase, and ∼0.5 nM PDE4D7-S129D and PDE4B1-
S133D to yield an initial rate of approximately −0.7 RFU/s. Less active
PDE4 enzymes were used at concentrations up to 4 nM. All data were
normalized relative to control wells lacking cAMP and are presented as
percent inhibition. An inhibitory concentration 50% (IC50) value was
calculated by fitting a sigmoidal dose response curve. Z′ quality factors
were routinely >0.6 for the assay.
4-Chloro-2-(3-chlorophenyl)-6-ethyl Pyrimidine (31). In a 1 L
round bottomed flask, 3-chlorobenzimidamide HCl 30(23.9 g, 154
mmol, 1 equiv), ethyl 3-oxopentanoate (27.8 g, 193 mmol, 1.25 equiv),
and ethanol (500 mL) were treated with NaOMe (10.0 g, 185 mmol,
1.20 equiv) and heated to reflux for 20 h. The resulting mixture was
cooled to rt and concentrated to ∼50 mL The slurry was cautiously
treated with 2 N HCl (219 mL), filtered, and the solid washed with H2O
and ether to afford crude 2-(3-chlorophenyl)-6-ethylpyrimidin-4(3H)-
one (9.3 g, 39% yield). The crude solid was treated with POCl3 (50 mL,
536 mmol, 13.4 equiv) at 0 °C and stirred at 100 °C for 5 h. After
cooling to rt, the mixture was added slowly to cold aq NaHCO3. The
mixture was extracted with EtOAc and the organics dried with Na2SO4,
and concentrated. The organic extract was dried over Na2SO4, filtered,
and concentrated under reduced pressure. Purification by silica gel
chromatography using DCM/EtOAc as eluent afforded 31 (9.3 g, 99%
yield) as a white solid. 1H NMR (CD3OD, 500 MHz) δ 8.32 (s, 1H),
8.28 (d, J = 7.5 Hz, 1H), 7.50−7.46 (m, 1H), 7.45−7.40 (m, 1H), 7.27
(s, 1H), 2.83 (q, J = 7.5 Hz, 2H), 1.34 (t, J = 7.5 Hz, 3H).
Synthesis of 4-Chloro-2-(3-hlorophenyl)-6,7-dihydro-5H-
cyclopenta[b]pyridine Hydrochloride (33). An 18 mL vial was
charged with 2,4-dichloro-6,7-dihydro-5H-cyclopenta[b]pyridine HCl
salt (32) (225 mg, 1 mmol), (3-chlorophenyl)boronic acid (165 mg,
1.06 mmol), Pd(Ph3P)4 (84 mg, 0.07 mmol), and K2CO3 (500 mg, 3.6
mmol). Toluene (6 mL), EtOH (2 mL) and water (2.5 mL) were
added, and the reaction was stirred under Ar at 90 °C until 32 was
consumed. The reaction was cooled to rt resulting in a biphasic system.
The organic phase was concentrated, and the crude material purified via
chromatography using hexanes/DCM (9:1 then 8:1) to afford 4-
chloro-2-(3-chlorophenyl)-6,7-dihydro-5H-cyclopenta[b]pyridine,
which was converted into its corresponding HCl salt by treating with 4
N HCl in dioxane (228 mg, 76% yield).
PDE4 Enzyme Crystallization and Structure Determination.
PDE4D with an N-terminal UCR2 fusion7 in 10 mM HEPES, pH 7.5,
100 mM NaCl, 1 mM dithiothreitol, 0.1 mM ZnCl2, and 0.1 mM
MgCl2, was crystallized at 5−10 mg/mL in the presence of 0.5 mM
compound (5, 6, or 28). Crystals were produced in a few days by sitting
drop vapor diffusion at 16 °C using a 1:1 mixture of protein:ligand to
crystallization condition containing 12.5% w/v PEG1,000, 12.5% w/v
PEG3,350, 12.5% w/v MPD, 0.03 M NPS (sodium nitrate, disodium
hydrogen phosphate, ammonium sulfate), and 0.1 mM MES/
Imidazole, pH 6.5. Crystals were harvested, flash frozen and shipped
Preparation of 4-Chloro-2-(3-chlorophenyl)-6-
(trifluoromethyl)pyridine (35). In a 500 mL round bottomed
flask, (3-chlorophenyl)boronic acid (1.59 g, 10.2 mmol), Pd(Ph3P)4
(0.608 g, 0.52 mmol), and K2CO3 (3.9 g, 27.8 mmol) in toluene (40
mL), EtOH (10 mL) and water (20 mL) were purged with Ar for 30
min. 34 (2.00 g, 9.26 mmol) was added, and purging continued for 10
N
DOI: 10.1021/acs.jmedchem.9b00193
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Journal of Medicinal Chemistry
Drug Annotation
to the listed synchrotron for screening and data collection. Data for 5
and 6 were collected at the Advanced Proton Source on Beamline 21-
ID-F equipped with a MAR Mosaic 300 and 225 CCD X-ray detector,
respectively. Data for 28 was collected at the Advanced Light Source on
Beamline 5.0.3 equipped with an ADSC Q315R CCD X-ray detector.
Diffraction data were reduced and scaled with XDS and XSCALE.72
Structures were solved by molecular replacement with previously
solved PDE4D structures and were refined using iterative cycles of
restrained refinement with Refmac573 and model-building using
COOT,74 both part of the CCP4 suite of programs.75,76 Structures
were validated by Molprobity77 ahead of deposition in the Protein Data
Bank.78,79 Diffraction data and refinement statistics are listed in
Supporting Information, Table 1.
Animal Experiments. Male ICR mice weighing 25−30 g were used
(Harlan, Indianapolis, IN) for all of the experiments. All behavioral tests
were performed during 9:30 am−16:30 pm and in accordance with the
“NIH Guide for the Care and Use of Laboratory Animals” (revised
2011) and were approved by the Institutional Animal Care and Use
Committee of the University at Buffalo. Compounds were dosed by per
os gavage (PO) in a vehicle of 5% DMSO, 5% Solutol in phosphate-
buffered saline (PBS). Rolipram was dosed at 1 mg/kg by intra-
peritoneal injection.
Novel Object Recognition. The novel object recognition test was
performed in a Plexiglas open field box (L30 cm, W50 cm, H40 cm) as
described elsewhere.80,81 Briefly, the task procedure consists of three
phases: habituation phase on day 1 for 10 min, training (T1) phase on
day 2 for 5 min, and testing (T2) phase on day 3 for 5 min. The duration
each animal spent exploring the objects was recorded. Time spent
exploring the identical objects in T1 was recorded as a1 and a2; time
spent exploring the familiar and the novel objects in T2 was recorded as
“a” and “b”, respectively. The following variables were calculated: e1 =
a1 + a2, e2 = a + b, the relative discrimination index d2 = (b − a)/e2.
Anesthesia Duration Test. Mice were dosed PO with the PDE4
inhibitor or vehicle or IP with rolipram; 30 or 15 min later (for PO and
IP doses, respectively), mice were injected IP with a mixture of
ketamine (80 mg/kg) and xylazine (10 mg/kg). The duration of
anesthesia was determined as the time between the loss and return of
the righting reflex and was used as an end point to measure the duration
of anesthesia.
ADME. Assays of kinetic solubility, liver microsome stability, CYP
inhibition using a fluorescent substrate, and cellular permeability in
MDCK-MDR1 cells were performed by Albany Molecular Research
Inc. using standard protocols. Metabolite identification was performed
by incubation with cryopreserved hepatocytes (0.5 × 106 cells/mL in
suspension) from male Wistar rats and human for 4 h at 37 °C. At the
end of the incubations, samples of the incubation medium were
analyzed by LC-UV/MS/MS analysis. Counter assays were performed
against PDE1−10 (BPS Biosciences), G-protein coupled receptors, ion
channels, and transporters (Cerep), and human cardiac ERG channel
(Eurofins Panlabs).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-616-224-0084. E-mail: mark@tetradiscovery.com.
ORCID
Janice A. Sindac: 0000-0003-2489-1576
Timothy J. Hagen: 0000-0003-1929-9121
Joel R. Walker: 0000-0001-6855-2969
Present Addresses
+For D.F.: UCB Pharma, 7869 NE Day Road West, Bainbridge
Island, Washington 98110, United States.
□For C.Z.: Janssen China R&D, 65 Guiqing Road, Building A,
Floor 6, Shanghai 200233, China.
●For R.F.C: The Walter Reed Army Institute of Research, 503
Robert Grant Avenue, Silver Spring, Maryland 20910, United
States.
Notes
The authors declare the following competing financial
interest(s): M.E.G, R.A.N., X.M., and J.A.S. are employees of
Tetra Discovery Partners, Inc. which has financial interest in
BPN14770.
ACKNOWLEDGMENTS
■
This work was supported by a research grant from the National
Institutes of Health Blueprint Neurotherapeutics Program
through the National Institute of Neurological Disorders and
Stroke and the National Institute on Aging (grant no.
NS078034) to M.E.G. with a subaward to Y.X. and
independently by Tetra Discovery Partners, Inc. We thank Dr.
Chuck Cywin, Dr. Enrique Michelotti, and Anirudh Chowdhary
for their contributions to the project.
ABBREVIATIONS USED
■
MeOH, methanol; DCM, dichloromethane; EtOAc, ethyl
acetate; EtOH, ethanol; NMP, N-methyl-2-pyrrolidinone;
ACN, acetonitrile; THF, tetrahydrofuran; DMF, N,N-dimethyl-
formamide; AcOH, acetic acid; HCl, hydrochloric acid; TFA,
trifluoroacetic acid; NaHCO3, sodium bicarbonate; Na2CO3,
sodium carbonate; K2CO3, potassium carbonate; Na2SO4,
sodium sulfate; MgSO4, magnesium sulfate; NH4Cl, ammonium
chloride; LiCl, lithium chloride; NaCl, sodium chloride;
NH4OH, ammonium hydroxide; LAH, lithium aluminum
hydride; POCl3, phosphoryl chloride; LiOH·H2O, lithium
hydroxide monohydrate; KCl, potassium chloride; DIEA,
d i i s o p r o p y l e t h y l a m i n e ; P d ( P P h 3 ) 4 , t e t r a k i s -
(triphenylphosphine)palladium (0); Pd(dppf)Cl2, ([1,1′- bis-
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b00193.
(diphenylphosphino)ferrocene]dichloropalladium(II)); BH ·
3
DMS, borane dimethyl sulfide complex; TFA, trifluoroacetic
acid; H3PO4, phosphoric acid; conc H2SO4, concentrated
sulfuric acid; Ar, argon; ACRDYS2, acrodysostosis type 1 with
or without hormone resistance; UCR, upstream conserved
regions; GWAS, genome-wide association studies; NMDA-R,
N-methyl D-aspartate receptor; BDNF, brain-derived neuro-
trophic factor; PDE4D7-S129D, dimeric isoform of PDE4D that
contains a UCR1 mutation (S129D) that mimics PKA
phosphorylation; PDE4B1−S133D, dimeric isoform of
PDE4B that also contains a UCR1 mutation (S133D) that
mimics PKA phosphorylation; PDE4D7-S129(wt), native
isoform of PDE4D7 that lacks the UCR1 phosphorylation
mimetic mutation; FaSSIF, fasted state simulated intestinal fluid
X-ray diffraction data and refinement statistics, compar-
ison of 28 in vitro potency against PDE4D7-S129D using
the coupled enzyme assay of cAMP hydrolysis versus the
BPS Bioscience PDE Assay Kit Catalogue, binding pose of
apremilast in the PDE4 active site (PDF)
Molecular formula strings (CSV)
Accession Codes
Protein Structure Database: Compound 5, PDB 6NJI;
compound 6, PDB 6NJH; compound 28, PDB 6NJJ. Authors
will release the atomic coordinates and experimental data upon
article publication.
O
DOI: 10.1021/acs.jmedchem.9b00193
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Journal of Medicinal Chemistry
Drug Annotation
(16) Lynch, D. C.; Dyment, D. A.; Huang, L.; Nikkel, S. M.; Lacombe,
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P.; Consortium, F. C.; Parboosingh, J. S.; Innes, A. M. Identification of
novel mutations confirms Pde4d as a major gene causing
acrodysostosis. Hum. Mutat. 2013, 34 (1), 97−102.
(17) Lindstrand, A.; Grigelioniene, G.; Nilsson, D.; Pettersson, M.;
Hofmeister, W.; Anderlid, B. M.; Kant, S. G.; Ruivenkamp, C. A.;
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Robinson, K.; Taylan, F.; Wincent, J.; Laurell, T.; Pekkinen, M.;
Nordenskjold, M.; Makitie, O.; Nordgren, A. Different mutations in
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(18) Hoppmann, J.; Gesing, J.; Silve, C.; Leroy, C.; Bertsche, A.;
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in a family with acrodysostosis type 2 caused by a novel PDE4D
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