Synthesis and SAR Studies of 1 H-Pyrrolo[2,3- b]pyridine-2-carboxamides as Phosphodiesterase 4B (PDE4B) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00369

Herein we report the synthesis, SAR, and biological evaluation of a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e., lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h was selective against a panel of CNS receptors and represents an excellent lead for further optimization and preclinical testing in the setting of CNS diseases.

Full text of DOI:10.1021/acsmedchemlett.9b00369

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Letter
Synthesis and SAR studies of 1H-pyrrolo[2,3-b]pyridine-2-
carboxamides as phosphodiesterase 4B (PDE4B) inhibitors
Anish K Vadukoot, Swagat Sharma, Christopher D Aretz, Sushil Kumar, Nagsen Gautam,
Yazen Alnouti, Amy L Aldrich, Cortney E. Heim, Tammy Kielian, and Corey R. Hopkins
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00369 • Publication Date (Web): 24 Jan 2020
Downloaded from pubs.acs.org on January 26, 2020
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Synthesis and SAR studies of 1H-pyrrolo[2,3-b]pyridine-2-
carboxamides as phosphodiesterase 4B (PDE4B) inhibitors
Anish K. Vadukoot1, Swagat Sharma1, Christopher D. Aretz1, Sushil Kumar1, Nagsen Gautam1, Yazen
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Alnouti1, Amy L. Aldrich2, Cortney E. Heim2, Tammy Kielian2, and Corey R. Hopkins1,*
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198
2Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE
68198.
KEYWORDS: 1H-pyrrolo[2,3-b]pyridine-2-carboxamide, PDE4B, phosphodiesterase 4, scaffold hopping.
ABSTRACT: Herein we report the synthesis, SAR, and biological evaluation of a series of 1H-pyrrolo[2,3-b]pyridine-2-
carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited
acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e.,
lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h, was selective against a panel of CNS receptors
and represents an excellent lead for further optimization and pre-clinical testing in the setting of CNS diseases.
Phosphodiesterases (PDEs) are a family of enzymes that
catalyze the hydrolysis of the secondary signal messengers,
cyclic adenosine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP). Both cAMP and cGMP are essential
components for cell signaling and are known to regulate the
function of airway smooth muscle, inflammatory cells, and
immune cells.1, 2 PDEs in mammals have been characterized
into at least 11 different families of isozymes (PDE1-PDE11)
that differ in their selectivity and specificity for the hydrolysis
of cAMP and cGMP.3 Among them PDE4, PDE7, and PDE8
are cAMP specific while PDE5, PDE6, and PDE9 are cGMP
specific.4 PDE4 is expressed predominantly in inflammatory
and immune cells including lymphocytes, macrophages,
eosinophils, and neutrophils where it tightly regulates the
intracellular concentrations of cAMP.2 The critical role cAMP
in regulating inflammatory and autoimmune processes makes
PDE4 an attractive target for treating inflammatory diseases
such as chronic obstructive pulmonary disorder (COPD),
asthma, rheumatoid arthritis, and psoriasis.2
addition to inflammatory processes, PDE4B has been
implicated in a number of other therapeutic roles, such as
cancer6, Alzheimer’s disease5, addiction7-9, and Parkinson’s
disease.10 The pan-PDE4 inhibitors such as roflumilast and
apremilast have been approved for the treatment of COPD and
psoriatic arthritis. Rolipram, another non-selective PDE4
inhibitor, was developed as a potential treatment for depression
but its clinical use was abandoned due to a narrow therapeutic
index.11 However, these drugs have been hampered by dose
limiting gastrointestinal side effects, such as nausea and emesis.
These side effects are hypothesized to be partially associated
with the inhibition of PDE4D isoform.12 Hence there is an
urgent need for developing novel chemical scaffolds that are
brain penetrant and have preferential PDE4B binding. In this
paper, we describe the synthesis, docking studies, and
pharmacological and biological profile of a series of 1H-
pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective
and potent PDE4B inhibitors.
We first wanted to evaluate the 5,6-core structure of the
previously reported PDE4B inhibitor, 1 (PDE4B IC50 = 0.8
M).13 All of the synthesized compounds were initially
evaluated for their in vitro inhibitory activity against PDE4B
using rolipram as a positive control. Table 1 comprises the in
vitro results of inhibitory activity against PDE4B for the title
compounds under study, expressed as IC50 values and (%)
inhibition in parenthesis.
A number of variants were
synthesized (see Experimental Section) and evaluated in an
enzymatic PDE4B and PDE4D assay (BPS Biosciences, San
Diego, CA) for activity. Our aim of this initial SAR evaluation
was to systematically evaluate the nitrogen atom and their
placement within the 5,6-framework in order to get a better
understanding of the potency contributions of these atoms. The
first compound, pyrazolo[1,5-a]pyridine 2, showed some
activity against PDE4B, however, there was an ~5-fold loss of
Figure 1. Modifications of known PDE4B inhibitor 1, to new
compound 11h.
PDE4 exists in four known isoforms (PDE4A-D), among
which PDE4A, B, and D are widely expressed in the brain.5
Inhibition of PDE4 has been shown to have anti-inflammatory
and anti-psychotic effects in various preclinical models.2 In
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in Scheme 1. The final compounds 11a-o were synthesized
Page 2 of 8
potency compared to 1 (2, IC50 = 4.2 M). Replacing the
imidazole moiety with a thiophene (3, thieno[2,3-b]pyrazine)
led to an inactive compound. Removing the 6-membered ring
nitrogen to afford the 1H-benzo[d]imdazole 4 led to an ~3-fold
loss of activity (IC50 = ~2.4 M), and further deletion of the
imidazole nitrogen leaving the indole 5 led to an inactive
1
2
3
4
5
6
7
8
starting from the common intermediate 8, followed by Chan-
Lam coupling to yield 9.14 Next, saponification of the ester led
to the acid, 10, which was then reacted with a variety of amines
under standard coupling conditions (T3P, DIPEA) to yield the
final compounds 11a-o.15 The second set of targets, 14a-w,
were synthesized in a similar fashion, but with a reversal of the
steps. Initial saponification of the ester in 8, followed by amide
coupling (T3P, DIPEA) led to the penultimate compound 13.
Final targets, 14a-w, were isolated after Chan-Lam coupling
(Cu(OAc)2, pyridine).14
compound.
Interestingly, moving to the pyrrolo[2,3-
b]pyridine, 7, led to an increase in potency (PDE4B IC50 = 0.48
M). Lastly, the N-methylated pyrrolo[3,2-b]pyridine, 6, was
not active. This is an interesting result as compounds such as
this with a further cyclic ring between the pyrrole nitrogen and
the amide have been reported as potent PDE4B inhibitors.
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Table 1: Identification of the initial lead compound (1-7).
IC50, nM;
(% Inhibition @ 10 M)
Cmpd
1
Structure
PDE4B
800
PDE4D
~8,200
Scheme 1. Synthesis of the final target compounds.
In Table 2, we explored the substitution effects of the amide
portion of the 1H-pyrrolo[2,3-b]pyridine series of compounds
by holding the 3,4-dichlorophenyl northern portion constant. It
can be seen from the IC50 values that most of the 1H-
pyrrolo[2,3-b]pyridine derivatives showed moderate to good
inhibition against PDE4B (IC50 ranges from 0.11 – 1.1 µM, %
inhibition of 99 – 20% at 10 µM). Careful analysis of the SAR
(entries 11a-o) reveals the importance of ring size and extent of
hydrophobicity on the overall activity and selectivity against
PDE4B over PDE4D. Our first analogs explored secondary
amides with small aliphatic cyclic or methyl furan groups. The
cyclopropyl, 11a, and 3-fluorocyclobutane, 11c, both retained
activity against PDE4B (IC50 = 0.63 and 0.6 M, respectively),
and were selective for PDE4D (<50% inhibition at 10 M, the
term selective is applied to compound <50% inhibition). The
cyclobutyl, 11b, difluorocyclobutyl, 11d, as well as the
methylfuran, 11e, were all much less active or inactive. Moving
to a tertiary amide (cyclic amide) was a more productive
substitution pattern. By replacing the NH-cyclobutyl in 11b
with the direct azetidine, 11f, an ~10-fold increase in activity
against PDE4B (IC50 = 0.11 M) was observed. This increase
in PDE4B activity was also accompanied by an increase in
PDE4D activity (IC50 = 0.45 M). Addition of a single fluorine
(3-fluoroazetidine, 11g) or a difluorine (3,3-difluoroazetidine,
11h) also produced potent compounds with varying degrees of
selectivity against PDE4D. The mono-fluoro, 11g, showed
some selectivity against PDE4D (PDE4B IC50 = 0.23 M and
77% inhibition of PDE4D at 10 M), whereas the
difluoroazetidine was ~6-fold selective for PDE4B (PDE4B
IC50 = 0.14 M vs. PDE4D IC50 = 0.88 M). Elongation of the
tertiary amide by incorporation of a 3-trifluoromethyl group
also produced an active compound (11i, PDE4B IC50 = 0.43
M), but with only an ~2-fold selectivity versus PDE4D.
Further elaboration of the amide space with a series of
spirocyclic rings (entries 11j-m) only exhibited moderate to
weak inhibition against PDE4B. Introduction of a basic
nitrogen in the spirocyclic ring (entries 11n-o) lead to a
significant loss of activity against PDE4B and PDE4D. This
2
3
4,200
(46)
(21)
(21)
4
~2,400
~2,000
5
6
(58)
(56)
(31)
(27)
Cl
N
O
N
N
HN
Cl
7
480
110
(41)
110
N
O
HN
Rolipram
aPDE4 assays run at BPS Biosciences, San Diego, CA, n = 2.
Having identified the pyrrolo[2,3-b]pyridine, 7, as a
productive replacement, we further elaborated this ring system
in order to fully vet this ring system. The analogs in Tables 2
and 3 were synthesized from common intermediates as shown
2
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aPDE4 assays run at BPS Biosciences, San Diego, CA, n
= 2.
may be due to lower lipophilic binding compared to 11j and
1
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4
5
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7
8
unfavorable interactions due to steric hindrance in the catalytic
site of the enzyme. In this series, we were able to identify a
novel scaffold 11h bearing a 3,3-difluroazetidine ring, which
exhibited higher inhibitory activity (0.14 µM) and 6-fold
selectivity over PDE4D than its corresponding analogs. This
compound was comparable in activity to the standard
compound, rolipram, and was more selective versus PDE4D.
Based on the above results, we further evaluated a
combination of aryl and amide substituents on the 1H-
pyrrolo[2,3-b]pyridine ring system to develop more potent and
selective PDE4B inhibitors. The aryl groups were chosen based
on the activity in the originally reported series.13 In entries 14a-
c, when R was 3-chloro-4-methylphenyl, increasing the size of
the amide led to a decrease in activity by ~2-20 fold, similar to
Table 2. SAR with variations at the R1 group, 11a-o.
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that seen previously.
However, moving to the 3-
fluoroazetidine, 14d, did not produce the same improvement in
potency as above, but this seemed to be an anomaly. When
evaluating other R groups (4-chloro-3,5-difluorophenyl, 14f-i,
4-cyano-3,5-difluorophenyl, 14j-o, 4-chloro-3-fluorophenyl,
14s-v), PDE4B activity was improved by using the 3-
fluoroazetidine tertiary amide. In order to fully investigate the
effect of R substitution on the core ring, we explored a series of
heteroaryl groups while keeping R1 (3-fluoroazetidine) constant
as shown in entries 14w-z,bb, Table 3. These heteroaryl
substituents indicate that substituted pyridyl (entries 14x and
14z) retains activity against PDE4B (IC50 = 0.49 and 0.98 M,
respectively); although the activity is less than the 11h.
Interestingly, the 3-fluoro-4-methoxyphenyl group, 14y, lost
activity against PDE4B; however, the activity could be regained
by the addition of an additional fluorine (3,5-difluoro-4-
methoxyphenyl, 14bb, IC50 = 0.41 M). This compound also
showed ~15-fold selectivity versus PDE4D. To our surprise,
the 3,3-difluoroazetidine moiety (i.e., 11h) that provided the
best compound previously, was not a productive moiety in this
new group of compounds (e.g., 14e and others). Lastly, a
methylene spacer was introduced between the phenyl group and
the pyrrole (benzyl) and this change led to an inactive
compound (14w).
IC50, nM;
(% Inhibition @ 10 M)a
Cmpd
11a
R1
PDE4B
630
PDE4D
(59)
11b
11c
11d
1,100
600
(24)
(36)
(23)
(11.6)
11e
11f
11g
(70)
110
230
(52)
450
(77)
11h
140
880
Table 3: SAR with variations at both R and R1 group (14a-
w).
11i
11j
430
1,000
(39)
(73)
IC50, nM;
(% Inhibition @ 10
M)
11k
11l
(39)
(50)
(17)
(29)
Cmpd
14a
R
R1
PDE4B
480
PDE4D
(41)
14b
14c
2,300
9,400
(43)
11m
11n
980
~2,300
(6)
(25.6)
14d
910
(60)
(20)
14e
14f
(47)
(26)
(53)
11o
(33)
110
(13)
110
1,400
Rolipram
3
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14g
14h
1,300
210
(10.7)
14aa
1
2
3
4
5
6
7
(37)
410
(35)
(20)
(62)
14bb
~6,300
(25)
14i
(48)
(27)
14cc
8
9
14j
1,400
1,800
(58)
(18.5)
14dd
(72)
110
(58)
110
10
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14k
14l
(16.6)
(12.1)
(18)
Rolipram
aPDE4 assays run at BPS Biosciences, San Diego, CA, n = 2.
In order to gain an understanding of the structural basis for
the SAR observed, we also studied the binding of the
synthesized inhibitors through computational modeling. The
3D structure of the PDE4B was taken from a Protein Data Bank
entry (PDB: 4X0F) having the ligand rolipram in place.
Docking studies were performed using Maestro 11.0
(Schrödinger, Small-Molecule Drug Discovery Suite 2018-3)
software using PDE4B in complex with (R)-(–)-rolipram as the
starting point. The optimized ligands were docked using Glide
software in Maestro 11.0. The resulting structures were then
analyzed using pose viewer to identify specific contacts
between ligand and receptors. As shown in Figure 2 (top), the
primary SAR study and docking results suggested that entry
11h core ring shows π-π stacking interactions with residues
Phe618 and Tyr405. The azetidine ring extends into the
catalytic domain to block the access of cAMP thereby laying
the foundation for PDE4B inhibition. The docking results were
also consistent with the observed SAR studies. Smaller groups
like cyclopropyl/difluoroazetidine occupy the smaller pocket in
the catalytic domain while larger spirocyclic groups at this
position lead to steric hindrance and therefore unfavorable
interactions leading to significant loss of PDE4B inhibitory
activity.
14m
(47)
14n
14o
430
(66)
(27)
(41)
14p
14q
2,900
5,000
(10.1)
(28.2)
14r
14s
(47)
970
(25)
(24)
14t
1,300
610
(39)
(71)
(25)
(37)
14u
14v
14w
(48)
(67)
14x
490
1,600
14y
14z
(77)
980
(52)
~1,900
4
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Figure 3. Target compounds inhibit macrophage pro-
inflammatory activity. Bone marrow-derived macrophages
were unstimulated (-) or pre-treated with the indicated
concentrations of the well-characterized PDE4 inhibitor
rolipram or target compounds (1 or 10 µM) for 30 min prior to
LPS (100 ng/ml; A) or Pam3Cys (10 µg/ml; B) stimulation.
After a 24 h treatment period, conditioned medium was
collected to quantitate TNF-α expression by ELISA. Results are
reported as the mean values (+ SD) from 3 independent
replicates for each treatment and were repeated in two separate
experiments. *, p < 0.05; **, p < 0.01; #, p < 0.0001; One-way
ANOVA with Dunnett’s multiple comparisons post-hoc
analysis.
Figure 2. Predicted binding mode of pyrrolo[2,3-b]pyridine
inhibitor 11h in the active site of PDE4B structure (PDB code
4X0F).
Since PDE4 inhibitors have well-documented anti-
inflammatory properties2, we evaluated the ability of these
compounds to inhibit macrophage pro-inflammatory activity by
measuring the production of the classical pro-inflammatory
cytokine TNF- (Figure 3). Two well-characterized pro-
inflammatory stimuli were tested, namely lipopolysaccharide
(LPS) and a synthetic lipopeptide (Pam3Cys) that represent
prototypical bacterial ligands for Toll-like receptors 4 and 2,
respectively (TLR4 and TLR2).16 All of the test compounds
inhibited TNF- production by mouse bone marrow-derived
macrophages to the same extent, or better than, the well-
characterized PDE4 inhibitor, rolipram (Figure 3). A dose-
dependent response was observed with regard to LPS-induced
TNF- release, where higher concentrations were less effective
across the group of compounds (Figure 3A), a phenomenon
which has been reported with other PDE4 inhibitors.17 A similar
dose-response was observed with some compounds when
macrophages were stimulated with Pam3Cys (Figure 3B);
nevertheless, cytokine production was significantly reduced
across the compound class. None of the compounds elicited
TNF- production when tested alone (data not shown). It is
noted that the compounds are all equally active in this assay,
and this is an effect we are currently investigating. Collectively,
these findings support the biological action of the target
compounds, which model the anti-inflammatory actions of the
well-characterized PDE4 inhibitor rolipram.18
Finally, having identified new PDE4 inhibitors, we next
evaluated select compounds in a battery of Tier 1 in vitro
DMPK assays (Table 4). A number of compounds evaluated
displayed low (QH <25%) clearance in both human and mouse
microsomes (14x, 14z, 14bb) and several other compounds
displayed moderate clearance (14i, 14j) in microsomes (QH
<50%). In addition to showing low clearance, both pyridine
containing compounds (14x, 14z) displayed an excellent free
fraction profile. In fact, most of the compounds evaluated had
free fraction >3% in both species, with the exceptions of 11a
and 14a in mice plasma (<1%).
Table 4. In vitro DMPK of selected compounds.
Plasma
Protein
Binding
Intrinsic Clearance (mL/min/kg)a,b
(%fu)a,c
Human
Mouse
CLIN
T
Hum
an
Cmpd
1
CLHEP CLINT
CLHEP
56.2
57.3
83.8
47.4
Mouse
5.6
29.6
12.3
149.5
157.4
1213.7
100.0
8.5
2.6
3.9
3.8
11a
11f
11h
178.6 18.8
194.3 19.0
0.6
3.0
97.1
17.3
2.7
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C.R.H., T.K. drafted and corrected manuscript with input from all
11i
295.6 19.6
172.5 18.7
1142.5
551.4
104
83.5
2.3
2.2
1
2
3
4
5
6
7
authors. A.K.V., C.D.A. and S.S. performed the chemical
synthesis and C.R.H. oversaw the medicinal chemistry, target
selection and interpreted the biological data. A.K.V. performed the
14a
14i
77.4
1.1
0.6
33.3
31.5
12.5
12.6
48.2
11.3
23.5
4.9
5.1
molecular modeling.
A.L.A. and C.E.H. performed the
14j
<49.5
133.7
<49.5
<49.5
<31.9
53.8
9.6
macrophage cellular assays and T.K. oversaw the experiments.
S.K. and N.G. performed the PK studies. Y.A. oversaw all of the
PK experiments.
14x
14z
14bb
<23.1 <11
<23.1 <11
<23.1 <11
6.6
<31.9
<31.9
47.4
30.2
39.3
13.3
Funding Sources
8
9
C.R.H. thanks the UNMC College of Pharmacy for start-up funds.
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Stability in Human Hepatocyte S9 Fractions
ACKNOWLEDGMENT
t1/2 (min)
49.4
CLHEP (mL/min/kg)
12.6
ER
0.63
The authors would like to thank Q2 solutions for the in vitro DMPK
experiments, BPS Biosciences for the PDE4 in vitro experiments,
and Frontage Laboratories for the in vivo PK experiments. The
authors would also like to thank Prof. Daryl J. Murry for the high-
resolution mass spectrometry data.
11h
aIn vitro DMPK studies performed at Q2 Solutions,
Indianapolis, IN bIntrinsic and predicted hepatic clearance
based on experiments in liver microsomes. c%fu = % fraction
unbound
ABBREVIATIONS
PDE4, phosphodiesterase 4; Kp, total brain:plasma ratio, T3P,
propylphosphonic anhydride; DIPEA, diisopropylethyl amine,
DMF, dimethylformamide; AUC, area under the curve; TNF,
tumor necrosis factor; ELISA, enzyme-linked immunosorbent
assay; PK, pharmacokinetics; DMPK, drug metabolism and
pharmacokinetics.
Lastly, we profiled 11h against a wider panel of PDE
isoforms (see Supplemental Tables 1) in a radioligand binding
assay for % inhibition at 10 M. 11h was selective against all
of the isoforms tested (<50% inhibition), except PDE3B (87%
inhibiton. In addition, 11h was also tested in the NIMH
Psychoactive Drug Screening Program (PDSP), which tests
compounds against a wide panel of targets (see Suppl. Table
2).19 In this panel, 11h was not active (<50% inhibition) against
all receptors except 5-HT2C (59% inhibition) and 2 (70%
inhibition). However, follow-up IC50 determinations showed
11h was only weakly active (5-HT2c, IC50 >10 M and 2, IC50
= 7.8 M)
In summary, a scaffold-hopping experiment identified a
novel 1H-pyrrolo[2,3-b]pyridine-2-carboxamide series of
PDE4B inhibitors. The compounds display a range of PDE4B
inhibition and selectivity versus PDE4D. In addition, we have
shown using molecular modeling how these compounds
interact with the enzyme. We have also shown that our
compounds are equipotent with rolipram in cellular assays
inhibiting macrophage pro-inflammatory cytokine activity.
Finally, compounds also show good in vitro PK profiles.
Further optimization and in vivo activity are on-going and will
be reported in due course.
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ASSOCIATED CONTENT
Supporting Information
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AUTHOR INFORMATION
Corresponding Author
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corticostriatal and hippocampal circuits: clinical overview and
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*Phone: 1 402-559-9729. Fax: 1 402-559-5643. E-mail:
corey.hopkins@unmc.edu
Author Contributions
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Keywords: PDE4, phosphodiesterase, CNS diseases, pyrrolo[2,3-b]pyridine
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