Discovery and Early Clinical Development of Isobutyl 1-[8-Methoxy-5-(1-oxo-3 H-isobenzofuran-5-yl)-[1,2,4]triazolo[1,5- a]pyridin-2-yl]cyclopropanecarboxylate (LEO 39652), a Novel "dual-Soft" PDE4 Inhibitor for Topical Treatment of Atopic Dermatitis

Article abstract of DOI:10.1021/acs.jmedchem.0c00797

We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while being stable in human skin. Compound 40 was selected as a clinical candidate as it was potent and rapidly degraded by blood and liver to inactive metabolites and because in preclinical studies it showed high exposure at the target organ: the skin. Preclinical and clinical data are presented confirming the value of the dual-soft concept in reducing systemic exposure.

Full text of DOI:10.1021/acs.jmedchem.0c00797

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Drug Annotation
Discovery and Early Clinical Development of Isobutyl 1‑[8-Methoxy-
5-(1-oxo‑3H‑isobenzofuran-5-yl)-[1,2,4]triazolo[1,5‑a]pyridin-2-
yl]cyclopropanecarboxylate (LEO 39652), a Novel “Dual-Soft” PDE4
Inhibitor for Topical Treatment of Atopic Dermatitis
Jens Larsen, Maja Lambert, Henrik Pettersson, Thomas Vifian, Mogens Larsen, Anna Ollerstam,
Pontus Hegardt, Cecilia Eskilsson, Steen Laursen, Anders Soehoel, Tine Skak-Nielsen, Lene M. Hansen,
Nina Ø. Knudsen, Stefan Eirefelt, Morten D. Sørensen, Tatiana G. Stilou, and Simon F. Nielsen*
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ABSTRACT: We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side
effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while
being stable in human skin. Compound 40 was selected as a clinical candidate as it was potent and rapidly degraded by blood and
liver to inactive metabolites and because in preclinical studies it showed high exposure at the target organ: the skin. Preclinical and
clinical data are presented confirming the value of the dual-soft concept in reducing systemic exposure.
We aimed to develop a topical treatment for severe AD.
Topical treatment of severe atopic dermatitis frequently
requires application of drug to more than 30% of the body
surface, the total applied dose being much higher than what is
usually used for oral dosing. As the atopic dermatitis skin
barrier is highly compromised, it is probable that a significant
proportion of the drug will be absorbed.6 This could lead to
significant systemic exposure of the applied compound and
potential adverse effects such as nausea, vomiting, and diarrhea
observed after systemic treatment with PDE4 inhibitors.7
Previously we described the discovery of 1 (see Figure 1), a
“soft”8 PDE4 inhibitor designed to achieve high local skin
concentrations but low systemic exposure upon topical
application due to a high hepatic clearance.9,10 The compound
was proven to be efficacious in AD patients,11 but nevertheless,
the clinical development of 1 was discontinued in part due to
safety concerns related to the systemic exposure of metabolites
of the candidate drug. In most patients 1 was rapidly converted
INTRODUCTION
■
Atopic dermatitis (AD) is a chronic inflammatory skin disease
characterized by eczematous lesions and intense itching.1 Skin
barrier dysfunction and immune cell activation participate in
the complex pathophysiology of AD. The immune activation in
AD lesions is dominated by Th2 cytokines (IL4, IL5, IL13)
and Th22 (IL22), but the Th1 and Th17 cytokines also play a
role in more chronic AD.2 The disease is most prevalent during
early infancy and childhood, with a prevalence of up to 20% of
children and 3% in adults. Limited topical treatment options
exist, namely, topical corticosteroids (TCSs) and topical
calcineurin inhibitors (TCIs). Both TCSs and TCIs are
coupled to some side effects. Only recently crisaborole3,4 was
approved as the first topical PDE4 treatment for mild to
moderate AD. The enzyme phosphodiesterase (PDE) 4 is
found in most immune cells, and it breaks down the secondary
messenger cyclic adenosine monophosphate (cAMP). PDE4
inhibitors induce increased levels of cAMP in leukocytes which
are associated with suppression of a number of proinflamma-
tory cytokines and other inflammatory mediators such as
prostaglandin E2.2 Increased PDE activity has been observed
in leukocyte preparation of patients with AD,5 and this further
emphasizes the relevance of pharmacological PDE4 inter-
vention in AD.
Received: May 18, 2020
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© XXXX American Chemical Society
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Figure 1. Examples of PDE4 inhibitors known prior to the discovery of the new triazolopyridine scaffold described in this paper.
to inactive metabolites as predicted, but unfortunately in some
individuals a different metabolic pathway was observed
resulting in high levels of an active pyridine N-oxide
metabolite.
This publication describes our effort to further reduce the
systemic exposure upon topical administration and mitigate the
risk of producing active metabolites, an exercise that should
minimize the risk of PDE4-mediated systemic side effects. We
set out to design “dual-soft” PDE4 inhibitors, i.e., compounds
with even higher systemic clearance than that of 1, driven by
extrahepatic as well as hepatic metabolism of the drug.
The approach was to take advantage of esterase-mediated
nomination of our first topical PDE4 clinical development
candidate 1, we also explored chemical scaffolds with the
trialkoxyphenyl top-part replaced by a triazolopyridine core as
exemplified by compounds 2 and 3 (Figure 1).15 We learned
that for these keto-linked triazolopyridine compounds the
cyclopropyl group of compound 3 is very important for PDE4
inhibition. Indeed, we believe that it occupies the same
“lipophilic pocket” in PDE4 as the cyclopropyl of roflumilast 4
(Figure 2).16
extrahepatic compound degradation in, for example, blood.
The major classes of human esterases are the carboxylesterases
(CES1 and CES2), paraoxonases (PON1, PON2, and PON3),
butyrylcholinesterase (BChE), and acetylcholineesterase
(AChE).12 Despite the fact that tissue distribution in humans
is not fully described for all esterases, some differences in
esterase expression in for instance skin versus blood have been
reported.13 Therefore, it was speculated that it should be
possible to identify ester-containing compounds suitable for
topical delivery,14 compounds that would be sufficiently stable
in skin to provide the desired pharmacological effect but at the
same time rapidly degraded both in blood and by the liver. The
dual elimination mechanism should ensure very fast clearance
and at the same time mitigate the risk of interpersonal
variations in drug levels due to polymorphism in esterases (and
other metabolizing enzymes).12 By applying this dual
elimination strategy, we anticipated that the systemic levels
of parent and potential active metabolites would be minimized.
In this paper we report the discovery of a novel series of
PDE4 inhibitors and our efforts to maximize the systemic
clearance of these, using screening models of human skin and
blood stability together with stability in human liver micro-
somes to guide the design.
The catalytic binding pocket of the PDE4 enzyme contains
two metal ions, which previously have been shown to be
crucial for design of PDE4 inhibitors. The compounds shown
Ultimately, compound 40 was selected as a clinical
development candidate, and in this paper, we report an in-
depth characterization of its metabolism and pharmacokinetic
profile as well as the early clinical data obtained with this
compound.
Figure 2. Overlay of roflumilast and compound 3 docked in the
roflumilast structure 1XOQ.30 Compound 3 was docked in the enol
form previously described for compound 1.9 The figure was made
RESULTS AND DISCUSSION
■
Scaffold Exploration: Focus on in Vitro Potency.
During the lead optimization campaign that led to the
̈
using Maestro, Schrodinger, LLC, New York, NY, 2019.
B
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in Figure 1 are all examples of that, as they are believed to gain
significant affinity via water mediated interactions between
their pyridine nitrogens and one of these two metals ions (see
Figure 3).
compound, 9, with potency comparable to the unsubstituted
phenyl analogue 5. Again an increased size of the keto
substituent reduced the PDE4 activity even further (10 and 11
vs 9).
A key finding was the importance of the orientation of the
carbonyl group. Compound 12 with the carbonyl group
pointing upward is significantly weaker PDE4 inhibitor than 6,
whereas 13 with the carbonyl orientation downward
apparently locks the conformation in a much more favorable
manner, consistent with a good fit for 13 to a pharmacophore
model based on the docked conformation of 3 (Figure 4). In
contrast, no conformation of 12 is able to fit the
pharmacophore.
In addition, the regioisomeric cyclic ketone 14 possessed
PDE4 inhibition comparable to that of both 6 and 13, again
highlighting the importance of both size and orientation of the
keto group. We assumed that these compounds interact with
the target via the carbonyl lone pairs, and to further explore
and substantiate this hypothesis, the nitrile substituted
analogue 15 was synthesized. The cyano nitrogen lone pair
of 15 should in principle be able to pick up a similar
interaction as achieved by the carbonyl lone pairs of 6, 13, and
14. As comparable potencies were observed, we assume they
all make similar interactions.
A wider range of substituents on the lower aromatic ring
were evaluated, but in general, other than those analogues
presented in Table 1, they led to significantly reduced in vitro
potency.
Having identified a new scaffold and explored possible
substitution points on the bottom ring, our attention focused
on the top part of the molecule. Modeling, as well as in house
SAR previously generated on the keto-linked scaffold series
shown in Figure 1, had taught us that substituents are not
allowed on the two unsubstituted positions on the left-hand
side of the upper aromatic ring. Likewise, we had learned that a
methoxy group seems to be one of the most preferred
substituents on top. Hence substitution appeared to be
tolerated only in the 1-postion of the cyclopropylpropyl ring.
Many substituents were evaluated, and during this exercise
secondary amides with the carbonyl directly attached to the
cyclopropyl ring revealed themselves as some of the most
interesting analogues as exemplified by 16, with 10 times
greater potency than the unsubstituted analogue 15.
Optimization of Skin and Blood Stability. Having
established an understanding of the potency SAR, the attention
was shifted toward the in vitro PK of the compounds, focusing
in particular on optimizing the balance between blood
instability and skin stability. We employed an empirical
screening approach rather than utilizing individual recombi-
nant esterases.
For investigation of the instability in blood we measured
degradation in freshly drawn human whole blood. As 90% of
the upper part of human skin (the epidermis) consists of
keratinocytes, we rationalized that stability of compounds in
keratinocyte suspensions could function as a primary human
skin stability assay.17 The chemical stability was also evaluated
as certain classes of the ester-containing compounds turned
out to be chemically unstable under assay/physiological
conditions.
Figure 3. Binding mode of roflumilast taken from 1XOQ.30 Shown
are the water molecules coordinating the Mg2+ metal ion and the
water mediated interactions with the pyridine nitrogen of Roflumilast.
̈
The figure was made using Maestro, Schrodinger, LLC, New York,
NY, 2019.
Thus, it came as no surprise that removing this water
meditated interaction by substituting the pyridine ring with a
phenyl led to reduced potency. The potency of the phenyl-
substituted analogue of 1 dropped 10-fold compared to that of
1 (data not shown). In contrast, in our search for a new,
simplified scaffold we were pleasantly surprised that 5 (Table
1), despite removal of the chlorines, keto linker and no option
for interaction with the metal atoms, still showed promising
potency. It should be noted that the phenyl group in the
simpler compound 5 most likely will not occupy the same
position as the pyridine ring of 1.
On the basis of the interactions observed for roflumilast in
the crystal structure, we hypothesized that introduction of a
hydrogen bond acceptor in compound 5 could improve
potency by reestablishing interactions with the metal ion.
Pleasingly, introduction of an acetyl group in the 3-position of
the lower aromatic ring supported this hypothesis, with 6 being
more than 40 times more potent compared to 5. On the basis
of this finding, we decided to further explore the triazolopyr-
idine scaffold.
Interestingly a very steep potency SAR was revealed when
we evaluated analogues with different substitutions on the
lower aromatic ring. For instance, as exemplified by
compounds 7 and 8, increased bulk around the carbonyl in
the 3-position is not tolerated. Changing the attachment point
of the acetyl group from the 3- to the 4-position gave
For “soft” and “dual-soft” compounds to be attractive the
main metabolite(s) must have a significantly lower PDE4
activity than the parent as only this will minimize the risk of
potential, systemic, target-related side effects. This may be
C
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a
Table 1. Structure−Activity Relationship of Early Triazolopyridine Compounds
a
All data represent an average of n ≥ 3 test occasions; standard deviations are as shown.
difficult to achieve for non-ester “soft” compounds as liver
metabolism often results in small changes and the metabolites
can sometimes be as active or even more active than the parent
compound (as seen for the pyridine N-oxide of compound 1).9
By utilization of ester cleavage, inactive metabolites are
intuitively easier to obtain as ester cleavage results in a
significant change in properties (size, polarity, and charge) of
the compound. Despite this, loss of activity is not guaranteed
and we made a thorough evaluation of the ester metabolites
(alcohols or acids) along with the parent ester-containing
molecules. Our preferred profile was to achieve at least a 10-
fold decrease in potency upon hydrolysis.
Considering the SAR of the newly discovered scaffold, we
deemed that two regions seemed amenable for introduction of
a “dual-soft spot”: esters with the carbonyl attached directly to
the lower aromatic ring and esters linked to the cyclopropyl
group.
Strategy A: Esters in the Lower Part of the Molecule.
Due to the steep SAR described above, we decided to evaluate
only methyl esters and lactones with the carbonyl directly
attached to either the 3- or 4-position of the lower aromatic
ring. On the basis of modeling, it was reasonable to assume
that lactones would mimic the cyclic ketones and that the
interaction of the carbonyl group with the target should be
kept. Similarly, for the methyl esters we expected to obtain
Figure 4. Fit of 13 (shown in green) to five-point pharmacophore
model based on the docked binding model of 3 (shown in orange).
̈
The pharmacophore model and fit were done with Phase, Schrodinger
̈
Inc. The figure was made using Maestro, Schrodinger, LLC, New
York, NY, 2019.
D
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potency somewhat between that of the methyl and ethyl
ketones.
Table 3. Structure−Activity Relationships in the “Alcohol”
and “Acid” Series
The 3-Position. The 3-substituted methyl ester 17 was, as
predicted, not the most potent compound, but addition of
small lipophilic amides at the cyclopropyl ring did increase the
PDE4 activity to an acceptable level, as illustrated with the
isobutylamide substituted analogues, 18 shown in Table 2. The
a
Table 2. Esters on the Lower Aromatic Ring
a
All data represent an average of n ≥ 3 test occasions; standard
b
deviations are as shown. In vitro clearance is determined in human
liver microsomes, and degradation half-life is determined in fresh
human whole blood and human epidermal keratinocytes.
reestablished the lost PDE4 activity, but unfortunately, it also
increased the whole blood stability. Further optimization
attempts did not lead to compounds with the desired in vitro
profile.
The 4-Position. In line with the SAR of compounds 9−11,
compounds 21 and 22 with the methyl ester in the 4-position
had rather poor PDE4 activity. In addition, the putative acid
metabolites formed upon hydrolysis were found to be even
more potent than the parent esters in the biochemical enzyme
assay. Consequently the 4-substituted ester series was
abandoned.
The properties of the lactone 23 did appear rather attractive.
The compound was equipotent with the corresponding
carbocyclic analogue 14, and in contrast to lactones with the
carbonyl in the 3-position (19 and 20), the extra oxygen in the
ring was well tolerated. It was rapidly degraded both in whole
blood and by the liver and was stable in our primary skin
stability assay, and an acceptable decrease in PDE4 activity was
observed upon hydrolysis. A range of analogues substituted in
most cases with lipophilic amide functionalities, as exemplified
by compound 24, were as expected highly potent and, as
desired, highly unstable in whole blood as well. The potency
shifts observed upon hydrolysis were acceptable, but most
importantly, the introduction of the amide functionality
disappointingly led to an unacceptable and quite surprising
poor performance in our primary skin stability assay. Thus, in
the end we did not manage to identify any potential candidates
a
All data represent an average of n ≥ 3 test occasions; standard
b
deviations are as shown. Number in parentheses is the IC50 of the
putative metabolite formed upon hydrolysis. In vitro clearance is
determined in human liver microsomes, and degradation half-life is
determined in fresh human whole blood and human epidermal
keratinocytes.
c
putative acid metabolites were significantly less potent then the
parent ester. Unfortunately compounds from this scaffold
series with a variety of amide substituents turned out to be too
stable in human whole blood.
The lactone 19 showed a similar effect to the methyl ester,
being significantly less potent than the cyclic ketone 13.
Apparently, the extra oxygen makes an unfavorable interaction
with the target. On the positive side, 19 did meet our primary
criteria for both skin stability and whole blood instability.
Furthermore, hydrolysis of the lactone led to a metabolite with
reduced PDE4 activity. Again, substitution with small lipophilic
amides at the cyclopropyl ring, as illustrated with 20,
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Table 4. Diester Analogues in the Triazolopyridine Scaffold
a
b
All data represent an average of n ≥ 3 test occasions; standard deviations are as shown. In vitro clearance is determined in human liver
microsomes, and degradation half-life is determined in fresh human whole blood and human epidermal keratinocytes.
from this series, balancing blood and HLM instability with
keratinocyte stability proving too challenging. Compound 23
appeared to be the most promising compound, but lower than
desired HLM clearance in the end disqualified the compound.
Strategy B: Esters in the Upper Part of the Molecule.
The second option we explored was to introduce an ester in
the upper right-hand side of the molecule. Two subseries were
evaluated, the “alcohol” and the “acid” series (Table 3).
In the “alcohol” series, esters were based on the three
potential alcohol metabolites 25−27. From a potency
perspective the most promising metabolite appeared to be
25, being only moderately potent against PDE4 and even less
potent in the cell-based assay. Further extension of the chain
resulted in alcohols (26, 27) with higher than desired PDE4
inhibitor activity, and we were not able to optimize the
potency of the corresponding esters to achieve the >10-fold
difference that we had defined as our minimum target criteria
(data not shown). Consequently, we focused on ester
derivatives of 25 using both aliphatic and heteroaromatic
acids. They all showed properties similar to those of ester 28.
They were potent, had high liver instability, and were degraded
reasonable quickly in blood but had very low stability in
human keratinocytes. It is speculated that esters from the
“alcohol series” may be degraded by a different esterase
compared to the other compounds described in this paper and
that this esterase is present in both skin and blood at significant
levels.
We abandoned the “alcohol series” and focused our
attention toward the “acids”. Optimization of this series also
started with an evaluation of the potential metabolites, and
again, in line with what we observed with the “alcohols” the
acid metabolites became more potent the further the polar
(acid) functionality was away from the aromatic ring. On the
basis of the known PDE4 crystal structures, extending the polar
functionality places it closer to the solvent front of catalytic
enzyme pocket. Consequently, analogues with the carboxylic
acid attached directly to the cyclopropyl group (29) and to a
slighter extent the ones having a methylene spacer (30)
F
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seemed the most appealing to us from a potency point of view.
The ethylene linked metabolite 31 was on the other hand
considered less useful as the metabolite per se was quite active
and the prepared esters were only moderately more potent
than the acid 31 (data not shown).
Several ester derivatives of acid 29, like compound 32, were
synthesized. In general, these compounds were potent; they
had adequate skin stability and rapid turnover in HLM but
were simply too slowly degraded in blood.
Making esters of acid 30 resulted in compounds with profiles
quite like that of 32, the main difference being an undesired
and unexpected increase in HLM stability as well as a reduced
potency shift observed upon ester hydrolysis.
In conclusion, esters with the carbonyl directly attached to
the cyclopropyl group held the most promise with regard to
parent potency, the potency shift upon hydrolysis, skin
stability, and hepatic instability, but they lacked the desired
whole blood instability.
38 appeared to be phototoxic in vitro (3T3 assay), a property
incompatible with topical dosing, and compound 49 exhibited
genotoxicity in the GreenScreen19 in the absence of S9.
The profiling of compound 40 did not identify any particular
safety concerns. Selectivity screening against PDE1−11 was
performed for the parent compound as well as all possible
metabolites (50, 53, 54, 55, 72). All compounds were highly
selective against PDE4, and 40 showed no significant
difference in inhibition of the four PDE4 isoforms (A, 1.2
nM; B, 1.2 nM; C, 3.0 nM; D, 3.8 nM). No significant signals
were observed in a broad panel screen (kinases, GPCR, ion
channels, and phosphatases). The emetic potential was
evaluated in ferrets. Due to the extremely fast degradation in
ferrets also, low exposure of 40 was observed. The iv dosing of
0.25 mg/kg provided the highest exposure (30 nM), and no
signals of emesis were observed. Compound 40 was therefore
selected as a clinical development candidate. An in-depth
discussion of the data generated on this compound is provided
in the sections below, while its basic potency and HLM, whole
blood, and keratinocyte stabilities are summarized in Table 5
along with those of its putative metabolites.
Concluding on “strategy A” and “strategy B”, we found it
quite challenging to balance all of the desired properties in one
molecule, in particular, the need for high skin stability and
blood instability.
Strategy C: Esters in Both Parts of the Molecule. The
“alcohol series” and the analogues exemplified by compound
24 were discarded due to unacceptable skin instability. For the
rest of the compounds described so far, the main issue was too
slow degradation in blood. In a final attempt to obtain
compounds with the desired in vitro PK and potency profile,
we went all in, incorporating two ester functionalities in the
molecules. The 3,4-lactone, exemplified by compound 23, was
identified as the most promising from “strategy A”, and it was
therefore chosen as the preferred bottom part, as we hoped it
would ensure both potency and blood instability. Combining
this with a carboxylic ester directly attached to the cyclopropyl
group, we explored the impact of varying the ester alkyl group.
(Table 4).
Table 5. Structure−Activity Relationship of LEO 39652
(40) and Its Putative Metabolites
Even the simplest alkyl esters turned out to be active in this
series. An increase in the size of the substituent going from
methyl (33) to ethyl (34) to n-propyl (35) gradually increased
potency against PDE4, and larger lipophilic substituents like
cyclopentyl (43), cyclohexyl (44), and cycloheptyl (45)
resulted in even more potent but perhaps also overall less
attractive compounds. Esters with branched butyl substituents
(37−40) balanced the in vitro potency and PK properties quite
elegantly, whereas the closely related n-butyl and cyclobutyl
ester analogues (41 and 42) did not perform as well in our
primary skin stability assay. Finally, substituents with moderate
polarity also seemed to be well tolerated with respect to both
in vitro PK and potency, compounds 46−52 containing either
an ether or alcohol functionality being representative examples.
As polarity increased, a corresponding decrease in liver
metabolism was observed, although we still considered it
acceptable for a soft compound. In general compounds from
this series were all rapidly degraded in blood as well as by the
liver, and fortunately most of the compounds appeared to be
stable in our human keratinocytes assay.
a
All data represent an average of n ≥ 3 test occasions; standard
b
deviations are as shown. In vitro clearance is determined in human
liver microsomes, and degradation half-life is determined in fresh
human whole blood and human epidermal keratinocytes.
Confirmation of Binding Mode. To obtain further
insight into its binding mode, a crystal structure of 40
complexed to PDE4D was obtained (Figure 5A). As expected,
the lactone coordinates with one of metal ions, while the
triazolopyridine core positions the methoxy and cyclopropyl
groups of 40 in the expected lipophilic pockets. For the
cyclopropyl this is determined by residues Phe414, Met431,
and Phe446. In addition, the isobutyl group forms hydro-
On the basis of these data, three compounds, 38, 40, and 49,
were selected for further profiling toward a potential candidate
selection. The profiling included in vivo efficacy observed in the
mouse CHROXA model18 as well as a more thorough PK and
safety evaluation. Parent compounds and putative metabolites
were synthesized and tested for off-target effects and in
particular for metabolism and PK. Unexpectedly, compound
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phobic interactions with Met347 and Ile450. Overall, the
observed binding mode is very similar (Figure 5B) to that
previously observed for the binding of an analogue of LEO
29102 (1).9
Chemistry. The compounds described in this paper can be
obtained following the synthetic sequences outlined in Scheme
1.20,21
First, nitropyridine 56 was reduced under classic Pd/C-
catalyzed hydrogenation conditions to the amine-pyridine 57,
which was then N-aminated at the pyridine nitrogen using
either O-(4-nitrobenzoyl)hydroxylamine or in situ generated
O-mesitylensulfonylhydroxylamine. The obtained 4-nitroben-
zoate (58) or 2,4,6-trimethylbenzenesulfonate (59) 1,2-
diamino-3-methoxypyridinium salts are thermally unstable.
Thus, preferably, immediately after isolation they were reacted
with the aldehyde of choice to form the key triazolopyridine
core (60−62). The aldehydes are either commercially available
or obtained in a few steps from readily available starting
materials. NIS/BF3·2H2O-mediated iodination and, if required,
classic deprotection reactions provided the 5-iodo-substiuted
building blocks (63−65), which can then be coupled with the
Figure 5. (A) Crystal structure of 40 (shown in magenta) complexed
with PDE4D solved to 1.6 Å. Key residues are shown in ball-and-stick
representation. Residue numbering is as in ref 9. (B) Overlay of 40
with the previously determined binding mode of an analogue (shown
in green) of LEO 29102 (1).9 The figure was made using Maestro,
̈
Schrodinger, LLC, New York, NY, 2019.
a
Scheme 1. General Preparation of the Triazolopyridine Scaffold Series
a
Reagents and conditions: (a) 10% Pd/C, 3 atm of H2, EtOH, rt, 91%; (b) O-(4-nitrobenzoyl)hydroxylamine or O-mesitylensulfonyl-
hydroxylamine, DCM, rt, 88−91%; (c) aldehyde, dioxane, 90 °C, 76% (R = H), 88% (R = COOEt), 47% (R = CH2O-TBDPS); (d) TBAF, THF,
rt, 79%; (e) NIS, BF3·2H2O, 84% (R = H), 77% (R = COOEt); 52% (R = CH2OH); (f) aq LiOH, dioxane, rt, 98%; (g) MsCl, Et3N, DCM, 0 °C,
91%; (h) NaCN, DMF, 80 °C, 66%; (i) HCl, MeOH, 70 °C, 81%; (j) LiOH, THF−MeOH−water, 50 °C, 69%; (k) dimethyl malonate, NaH,
toluene, 110 °C, 68%; (l) 6 N HCl, 100 °C, 68%; (m) Sn2Me6, (PPh3)2Pd(OAc)2, toluene, 100 °C, 100%; (n) Ar−Br, Pd(PPh3)4, toluene, 100 °C;
(o) Ar-B(OR)2, Pd(PPh3)4, aq K2CO3, DME, 80 °C or Ar-B(OR)2, Pd2 dba3, PCy3, K3PO4, dioxane−water, 110−145 °C; (p) BH3·THF, THF, 0
°C to rt; (q) R′OH, EDAC·HCl, DMAP, DCM, rt or R′NH2, HATU or PYBROP, Et3N, DMF, rt; (r) R″COCl, DIPEA, DCE, rt.
H
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Drug Annotation
a
Scheme 2. Preparation of the Clinical PDE4 Inhibitor 40
a
Reagents and conditions: (a) 5% Pd/C, 3 atm of H2, EtOAc−MeOH, rt, 97%; (b) HOSA, DBU, MeOH, 40 °C; (c) dimethyl cyclopropane-1,1-
dicarboxylate, DBU, MeOH, 65 °C; (d) aq NaOH, rt; (e) aq H2SO4, rt, 49%; (f) NBS, DMF−MeCN, 60 °C, 66%; (g) bis(pinacolato)diborane,
Pd(dppf)Cl2, KOAc, dioxane, 90 °C, 76%; (h) Pd(dppf)Cl2, Et3N, dioxane−water, 80 °C; (i) aq citric acid, rt, 69%; (j) iso-BuI, K2CO3, DMF, 70
°C, 81%.
lower aromatic ring of interest using either Stille or, more
commonly, classic Suzuki methodology. Mesylation of 63
followed by alkylation, decarboxylation, and/or hydrolysis
allowed us to access the 5-iodo-substiuted building blocks 66
and 67 required to generate the metabolites of the “acid
series”, 30 and 31. A subsequent BH3·THF mediated reduction
of these produced the “alcohol series” metabolite counterparts
26 and 27. The esters and amides presented in this paper were
in most cases synthesized with the aid of standard coupling
reagents like EDAC and HATU, respectively.
After 40 was nominated as clinical development candidate, a
chemistry optimization effort led to the synthesis shown in
Scheme 2, allowing us to access multikilogram quantities of 40
with excellent purity (>99%) in six steps with an overall yield
of 13%.22
With the optimized procedure column chromatography and
isolation of energy rich 1,2-diamino-3-methoxypyridinium
intermediates are no longer required. The aldehyde and the
preferred N-amination reagent O-(4-nitrobenzoyl) hydroxyl-
amine used in the original synthesis of 40 (Scheme 1) are (just
like 57) commercially available. However, to keep the synthesis
cost-efficient, we did produce both reagents in three steps from
cheap, readily available starting materials.22 Thus, in practice
the optimization effort did shorten the reaction sequence with
eight steps.
Systemic PK and Metabolism. The “dual-soft” design
strategy added extra complexity to understanding both the
dermal and systemic PK and metabolism due to the two ester
moieties in 40 which are hydrolyzed first in blood and
subsequently by the liver and other extrahepatic tissues
expressing esterases. Therefore, stability and metabolism of
40 and the putative hydrolytic metabolites 53, 54, and 55 were
investigated in human, rat, minipig, and monkey blood as well
as cryopreserved hepatocytes with the purpose of identifying
preclinical species with a suitable exposure and metabolism
profile of 40 for preclinical safety studies as well as making
predictions of human PK.
The half-life of 40 in blood was found to be short in all
species evaluated, ranging from 2 to 19 min (Table 6).
Table 6. In Vitro Stability in Human, Minipig, Monkey, and
Rat Whole Blood
t1/2 (min)
compd
human WB
monkey WB
minipig WB
rat WB
40
53
54
55
19
5
10
2
34
>60
>60
>60
>60
>60
>60
nd
nd
>60
>60
>60
Metabolite identification studies revealed a clear difference
in the degradation pathways in blood in vitro, as lactone
hydrolysis of 40 rapidly forms 53 in human and monkey blood,
whereas in rat blood the isobutyl ester is rapidly hydrolyzed
leading to complete transformation to 54 (Scheme 3). In
minipig blood formation of both 53 and 54 occurs. In addition,
it was found that the monoacid 53 is stable in human, minipig,
and monkey blood whereas in rat blood it is hydrolyzed to
diacid 55. Both 54 and 55 are stable in blood from all species.
I
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Drug Annotation
Scheme 3. Metabolites Formed in Vitro in Human, Minipig, Monkey, and Rat Whole Blood
Scheme 4. Proposed Metabolic Pathway in Human, Monkey, Minipig, and Rat Hepatocytes
The observed species difference in blood stability fits well
with previously described difference in expression of CES1
which has been reported to hydrolyze esters with a large and
bulky acyl group and small alcohol. CES1 is highly expressed in
rat blood, and carboxyl esterases are known to be absent in
both human and monkey blood.23,24 The expression levels in
minipig blood are to the best of our knowledge unknown.
In addition to rapid metabolism in blood, rapid metabolism
by the liver was expected. Metabolite identification studies of
40 and 53 (the only metabolite formed in human whole
blood) were performed in human, monkey, minipig, and rat
hepatocytes using UPLC/TOF-MS analysis. MS-based struc-
ture elucidation followed by comparison to synthetic standards
showed that for 40, the expected metabolites 53, 54, and 55
were found in all species. In addition, two major oxidative
metabolites were identified in human and monkey hepatocytes,
where hydroxylation in the 2-position of the isobutyl side chain
led to the formation of 50 and its lactone-hydrolyzed
counterpart, 72 (Scheme 4). Compound 50 is PDE4 active
(IC50 = 15 nM) and requires attention in relation to
toxicological qualification and levels obtained in human
studies. Furthermore, as shown in Scheme 4 the lactone
hydrolysis from 40 to 53 was found to be reversible in
hepatocytes.
The stability of 40 was investigated in human, minipig, and
rat hepatocytes (monkey not tested) and confirmed the
expected high hepatic turnover in all species (Table 7). On the
basis of the low hepatocyte stability, 40 would be expected to
Table 7. In Vitro Stability in Human, Minipig, and Rat
a
Hepatocytes
CLH (mL min−1 kg−1
minipig Hep
)
compd
human Hep
rat Hep
40
53
54
55
50
72
17
13
5.2
<5.2
16
>26
21
<11
<10
>26
13
>63
51
<19
33
63
53
7.1
a
Predicted hepatic clearance (CLH) from hepatocytes calculated using
the well stirred model. For calculations, liver blood flow used in
human, minipig, and rat were 20, 28, and 80 mL min−1 kg−1,
respectively.
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Drug Annotation
Table 8. In Vivo Clearance and Metabolite Profile Observed after Intravenous Administration of 40 in Rat, Minipig, and
Cynomolgus Monkey
in vivo clearance of 40 (mL min−1 kg−1
)
human
monkey
minipig
rat
110 (predicted)
300 (n = 4)
200 (n = 4)
930 (n = 3)
systemic exposure after single dose of 40
human
monkey
minipig
rat
a
b
b
b
ratio to total AUC (%)
ratio to total AUC (%)
ratio to total AUC (%)
ratio to total AUC (%)
40
53
54
55
50
72
1
12
15
71
<LLOQ
1
4
6
15
55
23
<LLOQ
<LLOQ
6
36
45
13
0.1
26
55
14
nd
nd
1
a
b
Data from clinical study after topical administration. Preclinical in vivo studies with intravenous administration (rat) or intravenous infusion
(minipig and monkey).
have a human in vivo hepatic clearance close to 100% of LBF,
and with additional extrahepatic clearance driven by esterases,
this should give a total clearance greatly in excess of LBF. The
monoacid metabolite formed in human blood, 53, is further
metabolized by the liver in all species, whereas 54 and 55 have
low to medium turnover.
PK studies of 40 in rat, minipig, and monkey after
intravenous administration were performed and confirmed a
very rapid elimination of 40 in all three species (Table 8).
Total clearance exceeded liver blood flow 5- to 10-fold in all
species, thereby confirming a substantial contribution from
extrahepatic metabolism. On the basis of these results,
clearance in man was predicted to be 110 mL min−1 kg−1
using allometric scaling of total clearance, which would lead to
very rapid elimination once 40 enters systemic circulation and
thereby ensuring the low systemic levels needed to avoid any
side effects mediated by PDE4 inhibition.
Parent 40 and the metabolites observed in vitro were
quantified in vivo in monkey, minipig, and rat intravenous
infusion studies. All three species showed 53, 54, and 55 as
major metabolites, with 53 and 55 accounting for >65% of
AUC. 50 and 72, which were observed as major in vitro
metabolites in human and monkey hepatocytes, turned out to
be circulating only in monkey (at low levels), and they were
not quantifiable in minipig and rat.
The dual elimination mechanism by liver and extrahepatic
esterases, combined with the species differences in both blood
and liver, made prediction of the in vivo metabolite profile
uncertain. Therefore in vivo pharmacokinetics and metabolite
profiles were investigated thoroughly and early in the
preclinical development. To ensure all human metabolites
were safety tested, it was decided to use monkey in our
preclinical safety studies.
Table 9. Skin Biopsy Concentrations of 1, 40, and
Metabolites 53, 54, and 55 at 24 h after Dermal Application
̈
in Monoglyceride Cream Formulation in Gottingen
Minipigs (n = 2)
skin biopsy concentration (μM)
monoglyceride cream
(0.25%)
monoglyceride cream
compd metabolite
(0.10%)
1
26
24
2
<0.1
6
14
17
0.6
<0.1
3
40
53
54
55
h prior to biopsy sampling. The biopsy concentrations and
dermal PK profile of 40 appear similar to 1 at both doses with
total drug concentrations 24 h after dosing of 17 μM (0.1%)
and 24 μM (0.25%) for 40 and 14 μM (0.1%) and 26 μM
(0.25%) for 1 (Table 9). On the basis of these numbers, full
target engagement over 24 h should be achieved with both
compounds. While metabolism in the pig skin does occur,
metabolites constitute ≤25% of the total drug related exposure
at 24 h, suggesting that skin metabolism should not prevent 40
from achieving an efficacious concentration in the skin.
Clinical Results. On the basis of the preclinical PK, safety
and efficacy profile, it was decided to enter 40 in a phase 1
safety and PK study with topical administration of increasing
body surface areas (5−20%) of patients with atopic dermatitis.
The compound was suspended 0.25% in a lipid cream designed
treatment of atopic dermatitis.
The PK profile after dermal administration of 40 is shown in
Table 10. We observed very low systemic exposure of the
PDE4 active parent molecule (40), and at the same time we
observed significantly higher levels of the inactive metabolites
53, 54, and 55. In fact, the Cmax of the major metabolite, 55,
was 30 times higher than the Cmax of the parent, compound 40.
In addition, the observed human metabolite profile after
topical dosing was similar to that seen in the cynomolgus
monkey after subcutaneous dosing, supporting our choice of
tox species. The levels of the active hydroxylated metabolite
(50) were extremely low/not detectable. Overall the human
PK profile proves that the concept of a “dual-soft” PDE4
inhibitor was successful, and we do not foresee any PDE4-
related systemic side effects after topical application.
Dermal PK and Metabolism. As described previously,
human epidermal keratinocytes, which comprise ∼90% of the
viable epidermis, were used as a screening model for
compound optimization, and 40 turned out to be fully stable
under the assay conditions used. Thus, it was not expected to
see significant metabolism of 40 in the skin.
To confirm sufficient stability and distribution in the skin,
̈
40 was evaluated in vivo in female Gottingen minipigs and
compared to 1 to support selection of clinical formulation. 40
and 1 were dosed in monoglyceride cream at concentrations of
0.1 and 0.25% (both efficacious doses of 1) at 24, 4, 1, and 0.5
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Drug Annotation
became evident to us that it is indeed a very challenging task to
design inhibitors that meet all of the in vitro PK and potency
criteria of a “dual-soft” compound. The lactone moiety turned
out to be desirable in the structure, though in this case we had
to combine it with another ester to achieve the desired
properties.
The underlying hypothesis that there are differences in
esterase expression in skin vs blood was eventually validated as
compounds with various profiles could be prepared and the
final candidate 40 fulfilled the criteria of being stable in skin
and unstable in blood (and liver).
Table 10. Pharmacokinetic Profile Observed after Dermal
Application of 40
Cmax (ng/mL)
AUC0−24 (ng·h/mL)
PDE4
active
cyno
monkey
cyno
monkey
a
b
a
b
compd
human
human
40
53
54
55
50
72
yes
no
no
no
yes
no
46.0
749
71.7
407
<LLOQ
10.5
0.0659
0.299
0.355
1.89
21.8
932
208
1296
<LLOQ
19.4
0.458
4.18
5.18
24.7
0.029
0.270
Species differences in metabolism were observed, with only
the cynomolgus monkey fully resembling the human
metabolite profile. The dual elimination mechanism was
successful in delivering a drug candidate with in vivo clearance
exceeding LBF 5- to 10-fold in all species tested. Due to the
dual elimination mechanism (blood and liver) and the
significant species differences observed in metabolite pathways,
the human clearance and PK were predicted based on
allometric scaling of total clearance. Human clearance was
predicted to be 110 mL min−1 kg−1, and while clinical PK data
to confirm this are not available, it was evident in healthy
volunteer and AD patients that only very low levels of 40 could
be detected and that significantly higher levels of themetabo-
lites 53, 54, and 55 were observed after dermal administration.
The systemic PK was in line with expectations for a “dual-
soft” compound, and skin PK from biopsies indicated minimal
degradation of 40 in human skin. It was therefore somewhat
surprising that the clinical effect was only marginally different
from placebo. The reasons behind lacking clinical efficacy have
been investigated in depth, and our belief is that this is related
to low free drug exposure of the candidate, not predicted by
the biopsy sampling technique.26 We believe translational
biomarkers and more advanced methods such as dermal open
flow microperfusion or MALDI imaging which can determine
drug at site of the target are likely necessary to measure
relevant skin concentrations.26−28
a
b
Single subcutaneous dosing of 30 mg/kg. Topical application on
20% of body surface of AD patients with 16 mg of compound 40.
The efficacy of 40 in patients with atopic dermatitis was
investigated in a left/right setup, treating two similar lesions
with either placebo cream or 40 suspended 0.25% in a lipid
cream. Efficacy was observed in AD patients, especially after
the first week of treatment, but after 3 weeks of treatment the
efficacy of the applied drug was only marginally better than
placebo cream.25 We were concerned that the exposure of the
compound in human skin was too low to result in clinical
efficacy; however the dermal concentrations of 40 were
determined from skin biopsies, and an average total drug
concentration of 6 μM was measured, which should be
sufficient for clinical efficacy. These contradictory results led to
the initiation of a retrospective and in-depth investigation of
the topical delivery of 40 to the skin using freshly excised
human skin in dermal open flow microperfusion27 studies and
a comparison of dermal interstitial fluid concentrations to skin
biopsy concentrations and the response of cAMP as a
pharmacodynamic biomarker.26 The conclusion of this
investigation was that the unbound concentration of 40 in
dermal interstitial fluid was low (6.5 nM). This could be
explained partly by a higher than predicted level of skin
metabolism and partly by a poor penetration of 40 through the
skin even though stratum corneum was completely removed in
the study to simulate barrier impaired AD skin. Furthermore,
no or limited effect on the pharmacodynamic biomarker cAMP
was observed.26 These results, taken together with MALDI
imaging studies on other topically applied drugs,27 indicate
that skin biopsy concentrations can be subject to contami-
nation from, for example, residual formulation during sampling
or drug in follicles and therefore should be used with caution.
In the case of 40, the 6 μM skin biopsy drug concentration did
not reflect the drug available at the site of the target in the skin.
EXPERIMENTAL SECTION
■
Synthesis. Chemistry: General Methods and Compound
Characterization. All reagents and starting materials were obtained
from commercial suppliers and used without further purification
unless otherwise stated. The organic solvents used were usually
anhydrous, and the reactions were run under an atmosphere of
nitrogen or argon unless otherwise noted. Reaction progress was
monitored using a variety of LC instruments equipped with
electrospray positive ionization detectors.
1H nuclear magnetic resonance (NMR) spectra were recorded at
300, 400, or 600 MHz. Chemical shift values (δ, in ppm) are quoted
in the specified solvent relative to internal tetramethylsilane (δ =
0.00) or chloroform (δ = 7.25) standards. The value of a multiplet,
either defined (doublet (d), triplet (t), quartet (q)) or not (m), at the
approximate midpoint is given unless a range is quoted. br s indicates
a broad singlet. Chromatography was performed on Merck silica gel
60 (0.040−0.063 mm). Preparative HPLC/MS was performed on a
Waters APS system with the following parameters: acid prep HPLC;
column, Waters Sunfire C-18, 100 mm × 19 mm, 5 μm; solvent
system, A = water (0.1% formic acid) and B = acetonitrile (0.1%
formic acid); basic prep HPLC; column, Waters XBridge C-18, 100
mm × 19 mm, 5 μm; solvent system, A = 50 mM ammonium
hydrogen carbonate and B = acetonitrile; flow rate = 20 mL/min;
method (8 min), linear gradient methods going from 5, 15, 30, or 50%
B to 100% B in 6 min and staying at 100% B for another 2 min. The
fractions were collected based on ion traces of relevant ions and PDA
signal (240−400 nm). All compounds were analyzed by UPLC−MS.
CONCLUSION
■
PDE4 inhibition is an attractive target for topical treatment of
AD, and a “dual-soft” approach was investigated: PDE4
inhibitors with high local concentration in the skin and low
circulating systemic concentrations based on rapid degradation
in blood and the liver.
We employed an empirical screening approach for
optimization, using metabolism in human whole blood,
human liver microsomes, and human keratinocytes. This
turned out to be a powerful strategy, although it did leave some
unknowns related to esterase specificity of the compounds.
Even though some trends were observed, design of unstable
compounds turned out to be far more difficult than we initially
expected. We learned that the incorporation of an ester group
is absolutely no guarantee of instability in whole blood, and it
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High resolution chromatography was performed using the Waters
Acquity UPLC and a 2.1 mm × 50 mm C18 RP column (1.8 μm
particles). The mobile phases consisted of 0.1% formic acid in an
aqueous 10 mM ammonium acetate solution for buffer A and 0.1%
formic acid in acetonitrile for buffer B. A standard binary gradient was
used for the reverse phase chromatography. All compounds had an
estimated UV purity of >95% by UPLC−MS.
same procedure as the one outlined for 6, the only difference being
that (3-acetylphenyl)boronic acid was replaced by (3-oxoindan-5-
1
yl)boronic acid. UPLC−MS (ESI+) m/z 320.14 (MH+). H NMR
(400 MHz, DMSO-d6) δ 8.27−8.13 (m, 2H), 7.75 (dd, J = 1.0, 7.9
Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 3.99 (s,
3H), 3.23−3.14 (m, 2H), 2.78−2.69 (m, 2H), 2.15 (tt, J = 4.9, 8.2
Hz, 1H), 1.08−0.90 (m, 4H).
Synthesis of 2-Cyclopropyl-8-methoxy-5-phenyl-[1,2,4]triazolo-
[1,5-a]pyridine (5). See the preparation of compound 102 described
4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
indan-1-one (13). See the preparation of compound 162 described in
WO 2010/069322. UPLC−MS (ESI+) m/z 320.14 (MH+). 1H NMR
(300 MHz, CDCl3) δ 7.91 (dd, J = 1.1, 7.6 Hz, 1H), 7.75 (dd, J = 1.2,
7.3 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 6.84 (s, 2H), 4.08 (s, 3H),
3.07−2.99 (m, 2H), 2.74−2.67 (m, 2H), 2.19 (tt, J = 4.9, 8.3 Hz,
1H), 1.22−1.15 (m, 2H), 1.08−0.98 (m, 2H).
5-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
indan-1-one (14). See the preparation of compound 160 described in
WO 2010/069322. UPLC−MS (ESI+) m/z 320.14 (MH+). 1H NMR
(300 MHz, DMSO-d6) δ 8.16−8.09 (m, 1H), 7.96 (dd, J = 1.5, 8.2
Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.16 (d, J
= 8.1 Hz, 1H), 4.00 (s, 3H), 3.22−3.16 (m, 2H), 2.75−2.68 (m, 2H),
2.16 (tt, J = 5.0, 8.1 Hz, 1H), 1.08−0.93 (m, 4H).
3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
benzonitrile (15). See the preparation of compound 135 described in
WO 2010/069322. UPLC−MS (ESI+) m/z 291.13 (MH+). 1H NMR
(300 MHz, DMSO-d6) δ 8.40 (t, J = 1.7 Hz, 1H), 8.33−8.25 (m,
1H), 7.95 (dt, J = 1.3, 7.6 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.32 (d, J
= 8.1 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 4.00 (s, 3H), 2.18 (tt, J = 5.0,
8.1 Hz, 1H), 1.09−0.92 (m, 4H).
1
in WO 2010/069322. UPLC−MS (ESI+) m/z 266.13 (MH+). H
NMR (600 MHz, DMSO-d6) δ 7.94−7.88 (m, 2H), 7.58−7.51 (m,
2H), 7.50 (tt, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H),
3.98 (s, 3H), 2.15 (tt, J = 4.8, 8.2 Hz, 1H), 1.04−0.99 (m, 2H), 0.98−
0.94 (m, 2H).
1-[3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]ethanone (6). See the preparation of compound 101
described in WO 2010/069322. UPLC−MS (ESI+) m/z 308.14
1
(MH+). H NMR (300 MHz, CDCl3) δ 8.49 (t, J = 1.9 Hz, 1H),
8.18−8.11 (m, 1H), 8.07−8.01 (m, 1H), 7.62 (t, J = 7.8 Hz, 1H),
6.98 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 4.07 (s, 3H), 2.67
(s, 3H), 2.24 (tt, J = 5.0, 8.4 Hz, 1H), 1.25−1.17 (m, 2H), 1.08−0.99
(m, 2H).
1-[3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]propan-1-one (7). The title compound was prepared
following the same procedure as the one outlined for 6, the only
difference being that (3-acetylphenyl)boronic acid was replaced by (3-
propanoylphenyl)boronic acid. UPLC−MS (ESI+) m/z 322.16
(MH+). 1H NMR (400 MHz, DMSO-d6) δ 8.49 (t, J = 1.8 Hz,
1H), 8.17 (ddd, J = 1.1, 1.9, 7.8 Hz, 1H), 8.06 (dt, J = 1.3, 7.7 Hz,
1H), 7.69 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.2
Hz, 1H), 4.00 (s, 3H), 3.13 (q, J = 7.2 Hz, 2H), 2.15 (tt, J = 4.9, 8.2
Hz, 1H), 1.13 (t, J = 7.2 Hz, 3H), 1.06−1.00 (m, 2H), 1.00−0.94 (m,
2H).
1-[3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]butan-1-one (8). The title compound was prepared
following the same procedure as the one outlined for 6, the only
difference being that (3-acetylphenyl)boronic acid was replaced by (3-
butanoylphenyl)boronic acid. UPLC−MS (ESI+) m/z 336.17
(MH+). 1H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J = 1.8 Hz,
1H), 8.16 (dt, J = 1.4, 7.8 Hz, 1H), 8.06 (dt, J = 1.4, 7.9 Hz, 1H), 7.69
(t, J = 7.8 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H),
4.00 (s, 3H), 3.07 (t, J = 7.1 Hz, 2H), 2.15 (tt, J = 4.9, 8.2 Hz, 1H),
1.76−1.61 (m, 2H), 1.13−0.84 (m, 7H).
1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-
yl]-N-isobutyl-cyclopropanecarboxamide (16). See the preparation
of compound 279 following the “general procedure 4” described in
WO 2010/069322. UPLC−MS (ESI+) m/z 390.19 (MH+). 1H NMR
(300 MHz, DMSO-d6) δ 8.51 (t, J = 5.7 Hz, 1H), 8.41 (t, J = 1.7 Hz,
1H), 8.29−8.22 (m, 1H), 8.00−7.93 (m, 1H), 7.74 (t, J = 7.9 Hz,
1H), 7.38 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 4.04 (s, 3H),
3.00 (dd, J = 5.7, 6.8 Hz, 2H), 1.77−1.66 (m, 1H), 1.55−1.49 (m,
2H), 1.44−1.37 (m, 2H), 0.81 (d, J = 6.8 Hz, 6H).
Methyl 3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-5-yl)benzoate (17). See the preparation of compound 117
described in WO 2010/069322. UPLC−MS (ESI+) m/z 324.14
1
(MH+). H NMR (600 MHz, DMSO-d6) δ 8.51 (t, J = 1.8 Hz, 1H),
8.19 (dt, J = 1.5, 7.8 Hz, 1H), 8.07 (dt, J = 1.4, 7.8 Hz, 1H), 7.70 (t, J
= 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 4.00
(s, 3H), 3.90 (s, 3H), 2.15 (tt, J = 4.9, 8.3 Hz, 1H), 1.05−1.00 (m,
2H), 1.00−0.94 (m, 2H).
1-[4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]ethanone (9). The title compound was prepared following
the same procedure as the one outlined for 6, the only difference
being that (3-acetylphenyl)boronic acid was replaced by (4-
acetylphenyl)boronic acid. UPLC−MS (ESI+) m/z 308.14 (MH+).
1H NMR (600 MHz, DMSO-d6) δ 8.10 (s, 4H), 7.29 (d, J = 8.1 Hz,
3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
benzoic Acid (Putative Metabolite of 17). See the preparation of
compound 136 described in WO 2010/069322. UPLC−MS (ESI+)
1
m/z 310.12 (MH+). H NMR (300 MHz, DMSO-d6) δ 12.87 (br s,
1H), 8.48 (t, J = 1.8 Hz, 1H), 8.15 (dt, J = 1.5, 7.9 Hz, 1H), 8.05 (dt,
J = 1.4, 7.7 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H),
7.14 (d, J = 8.2 Hz, 1H), 4.00 (s, 3H), 2.16 (tt, J = 5.0, 8.1 Hz, 1H),
1.08−0.91 (m, 4H).
1H), 7.16 (d, J = 8.3 Hz, 1H), 4.00 (s, 3H), 2.65 (s, 3H), 2.16 (tt, J =
4.8, 8.2 Hz, 1H), 1.05−1.00 (m, 2H), 1.00−0.95 (m, 2H).
1-[4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]propan-1-one (10). The title compound was prepared
following the same procedure as the one outlined for 6, the only
difference being that (3-acetylphenyl)boronic acid was replaced by (4-
propanoylphenyl)boronic acid. UPLC−MS (ESI+) m/z 322.16
Methyl 3-[2-[1-(Isobutylcarbamoyl)cyclopropyl]-8-methoxy-
[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoate (18). The title compound
was prepared following the “general procedure 2” described in WO
2010/069322. UPLC−MS (ESI+) m/z 423.20 (MH+). 1H NMR
(300 MHz, DMSO-d6) δ 8.65 (t, J = 5.7 Hz, 1H), 8.50 (t, J = 1.8 Hz,
1H), 8.18 (dt, J = 1.6, 7.7 Hz, 1H), 8.08 (dt, J = 1.7, 8.0 Hz, 1H), 7.70
(t, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H),
4.03 (s, 3H), 3.90 (s, 3H), 2.99 (dd, 2H), 1.71−1.60 (m, 1H), 1.57−
1.48 (m, 2H), 1.48−1.39 (m, 2H), 0.77 (d, J = 6.6 Hz, 6H).
3-[2-[1-(Isobutylcarbamoyl)cyclopropyl]-8-methoxy-[1,2,4]-
triazolo[1,5-a]pyridin-5-yl]benzoic Acid (Putative Metabolite of
18). A screw cap vial charged with 18 (25 mg, 0.060 mmol), water
(200 μL), dioxane (400 μL), and 2 M aqueous NaOH (500 μL) was
shaken at 40 °C for 10 min. Acidic prep HPLC purification afforded
the title compound. UPLC−MS (ESI+) m/z 409.19 (MH+). 1H
NMR (600 MHz, DMSO-d6) δ 13.19 (br s, 1H), 8.67 (t, J = 5.7 Hz,
1H), 8.48 (t, J = 1.8 Hz, 1H), 8.15 (dt, J = 1.6, 7.7 Hz, 1H), 8.06 (dt,
J = 1.5, 7.7 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H),
1
(MH+). H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 4H), 7.28 (d, J
= 8.1 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 4.00 (s, 3H), 3.12 (q, J = 7.2
Hz, 2H), 2.16 (tt, J = 4.9, 8.2 Hz, 1H), 1.13 (t, J = 7.2 Hz, 3H), 1.07−
0.92 (m, 4H).
1-[4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-
yl)phenyl]butan-1-one (11). The title compound was prepared
following the same procedure as the one outlined for 6, the only
difference being that (3-acetylphenyl)boronic acid was replaced by (4-
butanoylphenyl)boronic acid. UPLC−MS (ESI+) m/z 336.17
1
(MH+). H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 4H), 7.29 (d, J
= 8.1 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 4.00 (s, 3H), 3.06 (t, J = 7.1
Hz, 2H), 2.16 (tt, J = 4.9, 8.2 Hz, 1H), 1.75−1.61 (m, 2H), 1.12−
0.86 (m, 7H).
6-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
indan-1-one (12). The title compound was prepared following the
M
https://dx.doi.org/10.1021/acs.jmedchem.0c00797
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
7.23 (d, J = 8.1 Hz, 1H), 4.03 (s, 3H), 2.99 (dd, J = 5.7, 6.8 Hz, 2H),
1.70−1.60 (m, 1H), 1.56−1.48 (m, 2H), 1.47−1.38 (m, 2H), 0.77 (d,
J = 6.7 Hz, 6H).
4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
3H-isobenzofuran-1-one (19). See the preparation of compound 170
described in WO 2010/069322. UPLC−MS (ESI+) m/z 322.12
(MH+). 1H NMR (300 MHz, DMSO-d6) δ 8.12 (dd, J = 1.0, 7.6 Hz,
1H), 8.02 (dd, J = 1.0, 7.6 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.25 (d, J
= 8.1 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.47 (s, 2H), 4.01 (s, 3H),
2.15 (tt, J = 4.9, 8.2 Hz, 1H), 1.07−0.89 (m, 4H).
3-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-2-
(hydroxymethyl)benzoic Acid (Putative Metabolite of 19). A screw
cap vial charged with 19 (19 mg, 0.060 mmol), water (200 μL),
dioxane (400 μL), and 2 M aqueous NaOH (500 μL) was shaken at
40 °C until completion of hydrolysis. Bacic prep HPLC purification
afforded the title compound. UPLC−MS (ESI+) m/z 340.13 (MH+).
1H NMR (300 MHz, DMSO-d6) δ 7.80 (dd, J = 2.1, 7.3 Hz, 1H),
7.36−7.25 (m, 2H), 7.13 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz,
1H), 6.05 (br s, 1H), 4.40 (br s, 1H), 3.99 (s, 3H), 3.86−3.65 (m,
2H), 2.14−1.99 (m, 1H), 1.04−0.78 (m, 4H).
1H), 8.12−8.03 (m, 4H), 7.28 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.1
Hz, 1H), 4.00 (s, 3H), 2.16 (tt, J = 5.0, 8.2 Hz, 1H), 1.09−0.91 (m,
4H).
Methyl 4-[2-[1-(Isobutylcarbamoyl)cyclopropyl]-8-methoxy-
[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoate (22). The title compound
was prepared following the “general procedure 2” described in WO
2010/069322. UPLC−MS (ESI+) m/z 423.20 (MH+). 1H NMR
(300 MHz, DMSO-d6) δ 8.57 (t, J = 5.4 Hz, 1H), 8.13−8.03 (m, 4H),
7.37 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 4.04 (s, 3H), 3.91
(s, 3H), 2.99 (dd, J = 5.6, 6.8 Hz, 2H), 1.81−1.62 (m, 1H), 1.55−
1.47 (m, 2H), 1.46−1.37 (m, 2H), 0.81 (d, J = 6.8 Hz, 6H).
4-[2-[1-(Isobutylcarbamoyl)cyclopropyl]-8-methoxy-[1,2,4]-
triazolo[1,5-a]pyridin-5-yl]benzoic Acid (Putative Metabolite of
22). A screw cap vial charged with compound 22 (15 mg, 0.036
mmol), water (120 μL), dioxane (240 μL), and 2 M aqueous NaOH
(500 μL) was shaken at rt for 1 h. Acidic prep HPLC purification
afforded the title compound. UPLC−MS (ESI+) m/z 409.19 (MH+).
1H NMR (600 MHz, DMSO-d6) δ 13.12 (br s, 1H), 8.58 (t, J = 5.6
Hz, 1H), 8.07 (s, 4H), 7.36 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.3 Hz,
1H), 4.03 (s, 3H), 2.99 (dd, J = 5.7, 6.7 Hz, 2H), 1.69 (dh, J = 6.7,
13.4 Hz, 1H), 1.54−1.46 (m, 2H), 1.46−1.38 (m, 2H), 0.81 (d, J =
6.7 Hz, 6H).
5-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
3H-isobenzofuran-1-one (23). See the preparation of compound 167
described in WO 2010/069322. UPLC−MS (ESI+) m/z 322.12
(MH+). 1H NMR (300 MHz, CDCl3) δ 8.19−8.14 (m, 1H), 8.05 (d,
J = 8.0 Hz, 1H), 8.02−7.96 (m, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.86
(d, J = 8.1 Hz, 1H), 5.43 (s, 2H), 4.08 (s, 3H), 2.24 (tt, J = 4.9, 8.4
Hz, 1H), 1.25−1.17 (m, 2H), 1.10−1.01 (m, 2H).
N-Isobutyl-1-[8-methoxy-5-(1-oxo-3H-isobenzofuran-4-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide (20).
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-isobenzofuran-1-
one was synthesized as described in “preparation 14” in WO 2010/
069322 using 4-bromophthalide as the starting material instead of 4-
bromoindan-1-one. 1H NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 1.2,
7.2 Hz, 1H), 8.00 (dd, J = 1.1, 7.7 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H),
5.45 (s, 2H), 1.36 (s, 12H).
A vial charged with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
3H-isobenzofuran-1-one (1.63 g, 6.27 mmol), 1-(5-iodo-8-methoxy-
[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxylic acid (65)
(aka compound 306 described in WO 2010/069322) (1.50 g, 4.18
mmol), K3PO4 (3.10 g, 14.6 mmol, Pd2(dba)3 (76.5 mg, 0.0835
mmol), and PCy3 (46.9 mg, 0.167 mmol) in a degassed 2:1 dioxane−
water mixture (15 mL) was heated in a microwave oven at 130 °C for
10 min. The crude reaction mixture was then poured into 0.4 M
aqueous NaOH (220 mL). The aqueous phase was washed with
EtOAc (2 × 40 mL) before it was acidified with 4 M HCl and
extracted with EtOAc (3 × 50 mL). The combined organic phases
were dried over MgSO4, filtered, and evaporated to dryness. The
obtained solid was suspended in hot MeOH. Filtration afforded 1-[8-
methoxy-5-(1-oxo-3H-isobenzofuran-4-yl)-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl]cyclopropanecarboxylic acid. 1H NMR (300 MHz,
DMSO-d6) δ 12.55 (br s, 1H), 8.10 (dd, J = 1.1, 7.6 Hz, 1H), 8.02
(dd, J = 1.0, 7.6 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.0 Hz,
1H), 7.21 (d, J = 8.1 Hz, 1H), 5.49 (s, 2H), 4.04 (s, 3H), 2.07 (s,
1H), 1.57−1.46 (m, 2H), 1.46−1.35 (m, 2H).
4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-2-
(hydroxymethyl)benzoic Acid (Putative Metabolite of 23). A screw
cap vial charged with compound 23 (28 mg, 0.087 mmol), MeOH
(0.5 mL), and 2 M aqueous NaOH (0.5 mL) was shaken at rt for 2 h.
Basic prep HPLC purification afforded the title compound. UPLC−
1
MS (ESI+) m/z 340.13 (MH+). H NMR (300 MHz, DMSO-d6) δ
7.88 (d, J = 8.0 Hz, 1H), 7.82−7.70 (m, 2H), 7.20−7.03 (m, 2H),
6.02 (br s, 1H), 4.58 (s, 2H), 2.17 (tt, J = 5.0, 8.0 Hz, 1H), 1.08−0.86
(m, 4H).
N-Isobutyl-1-[8-methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide (24).
The title compound was prepared following the “general procedure
3” described in WO 2010/069322. UPLC−MS (ESI+) m/z 421.19
1
(MH+). H NMR (300 MHz, DMSO-d6) δ 8.52 (t, J = 5.6 Hz, 1H),
8.21 (s, 1H), 8.14 (dd, J = 1.5, 8.0 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H),
7.39 (d, J = 8.2 Hz, 1H), 7.27 (d, J = 8.2 Hz, 1H), 5.50 (s, 2H), 4.05
(s, 3H), 2.98 (dd, J = 5.7, 6.8 Hz, 2H), 1.77−1.59 (m, 1H), 1.54−
1.46 (m, 2H), 1.45−1.37 (m, 2H), 0.79 (d, J = 6.7 Hz, 6H).
2-(Hydroxymethyl)-4-[2-[1-(isobutylcarbamoyl)cyclopropyl]-8-
methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoic Acid (Putative
Metabolite of 24). A screw cap vial charged with 24 (13 mg, 0.030
mmol), THF (0.5 mL), and 1 M aqueous LiOH (0.5 mL) was shaken
at rt for 2 h. Basic prep HPLC purification afforded the title
A vial charged with 1-[8-methoxy-5-(1-oxo-3H-isobenzofuran-4-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylic acid (10
mg, 0.027 mmol), PYBROP (19 mg, 0.041 mmol), TEA (11.4 μL,
0.082 mmol), isobutyl amine (6.0 mg, 0.082 mmol), and DMF (0.5
mL) was shaken at rt overnight. Acid prep HPLC purification
1
compound. UPLC−MS (ESI+) m/z 439.20 (MH+). H NMR (300
1
afforded compound 20. UPLC−MS (ESI+) m/z 421.19 (MH+). H
MHz, DMSO-d6) δ 12.77 (br s, 1H), 8.72 (t, J = 5.7 Hz, 1H), 8.21 (d,
J = 1.7 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.93 (dd, J = 1.9, 8.2 Hz,
1H), 7.32 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 5.30 (br s,
1H), 4.92 (s, 2H), 4.04 (s, 3H), 3.05−2.97 (m, 2H), 1.70 (dh, J = 6.6,
13.4 Hz, 1H), 1.57−1.49 (m, 2H), 1.48−1.40 (m, 2H), 0.79 (d, J =
6.6 Hz, 6H).
NMR (600 MHz, DMSO-d6) δ 8.40 (t, J = 5.8 Hz, 1H), 8.15 (dd, J =
1.1, 7.7 Hz, 1H), 8.03 (dd, J = 1.0, 7.7 Hz, 1H), 7.78 (t, J = 7.7 Hz,
1H), 7.34 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 5.45 (s, 2H),
4.05 (s, 3H), 2.94 (dd, J = 5.7, 6.8 Hz, 2H), 1.62 (dh, J = 6.7, 13.5 Hz,
1H), 1.51−1.46 (m, 2H), 1.40−1.34 (m, 2H), 0.73 (d, J = 6.6 Hz,
6H).
3-[2-[1-(Hydroxymethyl)cyclopropyl]-8-methoxy-[1,2,4]triazolo-
[1,5-a]pyridin-5-yl]benzonitrile (25). See the preparation of com-
pound 150 described in WO 2010/069322. UPLC−MS (ESI+) m/z
321.14 (MH+). 1H NMR (300 MHz, DMSO-d6) δ 8.45 (t, J = 1.6 Hz,
1H), 8.34 (dt, J = 1.4, 8.0 Hz, 1H), 7.95 (dt, J = 1.3, 7.7 Hz, 1H), 7.75
(t, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H),
4.60 (t, J = 5.8 Hz, 1H), 4.01 (s, 3H), 3.89 (d, J = 5.9 Hz, 2H), 1.13−
1.06 (m, 2H), 1.06−0.99 (m, 2H).
3-[2-[1-(2-Hydroxyethyl)cyclopropyl]-8-methoxy-[1,2,4]triazolo-
[1,5-a]pyridin-5-yl]benzonitrile (26). A suspension of compound 30
(20 mg, 58 μmol) in THF (1.0 mL) was cooled under an argon
atmosphere to −10 °C. BH3·THF (1 M in THF, 100 μL, 100 μmol)
Methyl 4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-5-yl)benzoate (21). See the preparation of compound 119
described in WO 2010/069322. UPLC−MS (ESI+) m/z 324.14
(MH+). 1H NMR (600 MHz, DMSO-d6) δ 8.13−8.08 (m, 4H), 7.30
(d, J = 8.1 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 4.00 (s, 3H), 3.91 (s,
3H), 2.16 (tt, J = 4.9, 8.3 Hz, 1H), 1.05−1.00 (m, 2H), 1.00−0.95
(m, 2H).
4-(2-Cyclopropyl-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-
benzoic Acid (Putative Metabolite of 21). See the preparation of
compound 140 described in WO 2010/069322. UPLC−MS (ESI+)
1
m/z 310.12 (MH+). H NMR (300 MHz, DMSO-d6) δ 13.11 (br s,
N
https://dx.doi.org/10.1021/acs.jmedchem.0c00797
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Drug Annotation
was added, and the mixture was stirred at 0 °C for 3 h before more
BH3·THF (1 M in THF, 100 μL, 100 μmol) was added. The mixture
was left stirring in a cool bath overnight, and the temperature reached
10 °C. More BH3·THF (1 M in THF, 300 μL, 300 μmol) was then
added, and the reaction mixture was allowed to reach rt. After 6 h at rt
basic prep HPLC purification afforded the title compound. UPLC−
Solid LiOH (1.8 g, 45 mmol) was added at rt to a solution of
methyl 2-[1-(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-
cyclopropyl]acetate (3.5 g, 9.0 mmol) in THF (20 mL), MeOH (7
mL), and water (3.5 mL). The mixture was heated at 50 °C for 2 h
before the solvents were removed under reduced pressure. Water was
added, and the pH of the crude reaction mixture was adjusted to 5
with 1 M HCl before the aqueous phase was extracted with EtOAc.
Column chromatography afforded 2-[1-(5-iodo-8-methoxy-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl)cyclopropyl]acetic acid (66) (2.3 g, 69%
1
MS (ESI+) m/z 335.15 (MH+). H NMR (300 MHz, DMSO-d6) δ
8.40 (t, J = 1.5 Hz, 1H), 8.33 (dt, J = 1.7, 8.0 Hz, 1H), 7.95 (dt, J =
1.5, 7.7 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H),
7.15 (d, J = 8.2 Hz, 1H), 4.42 (t, J = 5.2 Hz, 1H), 4.01 (s, 3H), 3.63
(td, J = 5.0, 7.2 Hz, 2H), 2.00 (t, J = 7.2 Hz, 2H), 1.19−1.06 (m, 2H),
1.02−0.88 (m, 2H).
1
yield). H NMR (400 MHz, DMSO-d6) δ 7.46 (d, J = 8.1 Hz, 1H),
6.88 (d, J = 8.2 Hz, 1H), 3.93 (s, 3H), 2.82 (s, 2H), 1.28−1.21 (m,
2H), 1.06−0.98 (m, 2H).
3-[2-[1-(3-Hydroxypropyl)cyclopropyl]-8-methoxy-[1,2,4]-
triazolo[1,5-a]pyridin-5-yl]benzonitrile (27). The title compound
was synthesized following the same procedure as the one described
for compound 26 the only difference being that 31 was used as
starting material instead of 30. UPLC−MS (ESI+) m/z 349.17
A vial charged with compound 66 (800 mg, 2.14 mmol), (3-
cyanophenyl)boronic acid (473 mg, 3.22 mmol), K3PO4 (1.59 g, 7.50
mmol), Pd2(dba)3 (39.3 mg, 0.0429 mmol), and PCy3 (24.1 mg,
0.0858 mmol) in a degassed 2:1 dioxane−water mixture (19.2 mL)
was heated in a microwave oven at 130 °C for 10 min. The crude
reaction mixture was then poured into 0.05 M aqueous NaOH (210
mL). The aqueous phase was washed with EtOAc (2 × 40 mL) after
which the pH was adjusted to 3 with 4 M HCl. The aqueous phase
was extracted with EtOAc (3 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to dryness.
Acidic prep HPLC purification afforded compound 30. UPLC−MS
1
(MH+). H NMR (300 MHz, DMSO-d6) δ 8.46 (t, J = 1.8 Hz, 1H),
8.34 (dt, J = 1.6, 8.2 Hz, 1H), 7.94 (dt, J = 1.6, 7.5 Hz, 1H), 7.74 (t, J
= 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 4.32
(t, J = 5.1 Hz, 1H), 4.01 (s, 3H), 3.43 (q, J = 6.1 Hz, 2H), 1.83 (dd, J
= 5.2, 10.3 Hz, 2H), 1.74−1.60 (m, 2H), 1.19−1.08 (m, 2H), 0.95−
0.82 (m, 2H).
[1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-
2-yl]cyclopropyl]methyl 2-methylpropanoate (28). A screw cap vial
charged with compound 25 (20 mg, 0.062 mmol), DIPEA (12 mg,
0.094 mmol), 2-methylpropanoyl chloride (9.8 μL, 0.094 mmol), and
DCE (0.5 mL) was shaken at rt overnight. Volatiles was removed
under reduced pressure after which acidic prep HPLC purification
afforded the title compound. UPLC−MS (ESI+) m/z 391.18 (MH+).
1H NMR (600 MHz, DMSO-d6) δ 8.42 (t, J = 1.8 Hz, 1H), 8.31 (dt, J
1
(ESI+) m/z 349.13 (MH+). H NMR (300 MHz, DMSO-d6) δ 8.42
(t, J = 1.8 Hz, 1H), 8.40−8.32 (m, 1H), 7.94 (dt, J = 1.5, 7.8 Hz, 1H),
7.72 (t, J = 7.9 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.3 Hz,
1H), 4.01 (s, 3H), 2.78 (s, 2H), 1.31−1.17 (m, 2H), 1.14−0.98 (m,
2H).
3-[1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl]cyclopropyl]propanoic Acid (31). Dimethyl malonate
(4.54 mL, 37.9 mmol) was slowly added to a suspension of NaH
(60%, 1.19 g, 37.9 mmol) in toluene (200 mL) at 0 °C. The mixture
was heated at reflux for 1.5 h before a solution of [1-(5-iodo-8-
methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]-
methylmethanesulfonate (see preparation of compound 30) (5.0 g,
11.8 mmol) in toluene was slowly added at rt. The reaction mixture
was heated at reflux for an additional 3 h. Volatiles were removed
under reduced pressure, water was added, and the aqueous phase was
extracted with EtOAc. The organic phase was dried over anhydrous
Na2SO4. Column chromatography (eluent: 5% EtOAc in DCM)
afforded dimethyl 2-[[1-(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl)cyclopropyl]methyl]propanedioate (3.7 g, 68% yield).
A mixture of dimethyl 2-[[1-(5-iodo-8-methoxy-[1,2,4]triazolo-
[1,5-a]pyridin-2-yl)cyclopropyl]methyl]propanedioate (3.78 g, 8.06
mmol) in 6 N HCl (40 mL, 240 mmol) was heated at 100 °C for 16 h
before it was cooled to 0 °C. Water was added, and the aqueous phase
was extracted with EtOAc. The organic phase was dried over
anhydrous Na2SO4. Column chromatography (eluent: 3% MeOH in
CHCl3) afforded 3-[1-(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]-
= 1.6, 8.1 Hz, 1H), 7.95 (dt, J = 1.4, 7.7 Hz, 1H), 7.72 (t, J = 7.9 Hz,
1H), 7.38 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 4.45 (s, 2H),
4.01 (s, 3H), 2.51−2.37 (m, 1H), 1.32−1.26 (m, 2H), 1.19−1.14 (m,
2H), 0.97 (d, J = 7.0 Hz, 6H).
1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-
yl]cyclopropanecarboxylic Acid (29). See the preparation of
compound 191 described in WO 2010/069322. UPLC−MS (ESI+)
1
m/z 335.12 (MH+). H NMR (300 MHz, CDCl3) δ 8.12−8.07 (m,
2H), 7.78 (dt, J = 1.5, 7.9 Hz, 1H), 7.69−7.61 (m, 1H), 7.11 (d, J =
8.2 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 4.12 (s, 3H), 2.02−1.90 (m,
2H), 1.86−1.74 (m, 2H).
2-[1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl]cyclopropyl]acetic Acid (30). A mixture of [1-(5-iodo-8-
methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]methanol
(63) (prepared as compound 308 in WO 2010/069322) (4.0 g, 12
mmol), TEA (4.7 mL, 35 mmol), and MsCl (1.0 mL, 13 mmol) in
DCM (50 mL) was stirred at 0 °C for 15 min. Water was added, and
the reaction mixture was extracted with DCM. The combined organic
phases were dried over MgSO4, and evaporation to dryness afforded
[1-(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]-
methylmethanesulfonate (4.5 g, 91% yield) which was used in the
next step without further purification.
1
pyridin-2-yl)cyclopropyl]propanoic acid (67) (2.1 g, 68% yield). H
NMR (400 MHz, DMSO-d6) δ 12.04 (s, 1H), 7.47 (d, J = 8.1 Hz,
1H), 6.89 (d, J = 8.3 Hz, 1H), 3.93 (s, 3H), 2.58−2.52 (m, 2H),
2.14−1.98 (m, 2H), 1.22−1.11 (m, 2H), 1.02−0.90 (m, 2H).
A vial charged with compound 67 (800 mg, 2.01 mmol), (3-
cyanophenyl)boronic acid (455 mg, 3.10 mmol), K3PO4 (1.54 g, 7.23
mmol), Pd2(dba)3 (37.8 mg, 0.0413 mmol), and PCy3 (23.2 mg,
0.0827 mmol) in a degassed 2:1 dioxane−water mixture (19.2 mL)
was heated in a microwave oven at 130 °C for 10 min. The crude
reaction mixture was then poured into 0.05 M aqueous NaOH (210
mL). The aqueous phase was washed with EtOAc (2 × 40 mL) after
which the pH was adjusted to 3 with 4 M HCl. The aqueous phase
was extracted with EtOAc (3 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to dryness.
Acidic prep HPLC purification afforded compound 31. UPLC−MS
NaCN (825 mg, 16.8 mmol) was added slowly to a solution of [1-
(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]-
methylmethanesulfonate (3.7 g, 8.4 mmol) in DMF (20 mL). The
mixture was heated at 80 °C for 2 h. Water was added, and the
mixture was carefully extracted with EtOAc. The aqueous phase was
quenched with a solution of KMnO4. The combined organic phases
were dried over MgSO4, and evaporation to dryness afforded 2-[1-(5-
iodo-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]-
acetonitrile (2.0 g, 66% yield) which was used in the next step without
further purification.
A solution of 2-[1-(5-iodo-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl)cyclopropyl]acetonitrile (4.0 g, 11 mmol) and HCl in
MeOH was stirred for 8 h at 70 °C. The solvent was then removed.
Aqueous NaHCO3 was added, and the mixture was extracted with
EtOAc. Column chromatography afforded methyl 2-[1-(5-iodo-8-
methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropyl]acetate (3.5 g,
81% yield).
1
(ESI+) m/z 363.15 (MH+). H NMR (300 MHz, DMSO-d6) δ 8.41
(t, J = 1.7 Hz, 1H), 8.34 (dt, J = 1.5, 8.0 Hz, 1H), 7.94 (dt, J = 1.2, 7.7
Hz, 1H), 7.73 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.16 (d, J
= 8.3 Hz, 1H), 4.01 (s, 3H), 2.54−2.45 (m, 2H), 2.07−2.00 (m, 2H),
1.19−1.09 (m, 2H), 1.02−0.91 (m, 2H).
O
https://dx.doi.org/10.1021/acs.jmedchem.0c00797
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Isobutyl 1-[5-(3-Cyanophenyl)-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl]cyclopropanecarboxylate (32). A mixture of compound
29 (17 mg, 0.050 mmol), EDAC·HCl (19 mg, 0.10 mmol), DMAP
MHz, DMSO-d6) δ 8.22 (br s, 1H), 8.14 (dd, J = 1.5, 8.0 Hz, 1H),
7.99 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.3 Hz,
1H), 5.51 (s, 2H), 4.82−4.74 (m, 1H), 4.04 (s, 3H), 1.60−1.56 (m,
1H), 1.56−1.50 (m, 2H), 1.50−1.40 (m, 3H), 1.13 (d, J = 6.2 Hz,
3H), 0.78 (t, J = 7.4 Hz, 3H).
Isobutyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (40). See the
preparation of compound 3 described in WO 2013/092739. The
synthesis of compound 40 on kilogram scale is provided in WO 2014/
206903. UPLC−MS (ESI+) m/z 422.17 (MH+). 1H NMR (600
MHz, DMSO-d6) δ 8.22 (br s, 1H), 8.13 (dd, J = 1.5, 8.1 Hz, 1H),
8.00 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 8.2 Hz,
1H), 5.51 (s, 2H), 4.04 (s, 3H), 3.84 (d, J = 6.5 Hz, 2H), 1.85−1.74
(m, 1H), 1.61−1.54 (m, 2H), 1.54−1.46 (m, 2H), 0.78 (d, J = 6.7 Hz,
6H).
i
(12 mg, 0.10 mmol), BuOH (45 μL, 0.49 mmol) in DCM (1.0 mL)
was stirred at rt overnight. Volatiles were removed under reduced
pressure. Acidic prep HPLC purification afforded the title compound.
UPLC−MS (ESI+) m/z 391.18 (MH+). 1H NMR (300 MHz,
DMSO-d6) δ 8.40 (t, J = 1.9 Hz, 1H), 8.29 (dt, J = 1.4, 8.0 Hz, 1H),
7.96 (dt, J = 1.3, 7.7 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.41 (d, J = 8.1
Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 4.03 (s, 3H), 3.85 (d, J = 6.5 Hz,
2H), 1.95−1.67 (m, 1H), 1.62−1.54 (m, 2H), 1.54−1.46 (m, 2H),
0.79 (d, J = 6.8 Hz, 6H).
Methyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (33). Com-
pound 33 was synthesized following the same procedure as the one
outlined for compound 32, the only difference being that 54 was used
as starting material instead of 29. UPLC−MS (ESI+) m/z 380.13
(MH+). 1H NMR (600 MHz, DMSO-d6) δ 8.23 (br s, 1H), 8.14 (dd,
J = 1.5, 8.1 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz,
1H), 7.24 (d, J = 8.3 Hz, 1H), 5.52 (s, 2H), 4.04 (s, 3H), 3.62 (s,
3H), 1.59−1.53 (m, 2H), 1.53−1.47 (m, 2H).
Butyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (41). Com-
pound 41 was synthesized following the same procedure as the one
outlined for compound 33, the only difference being that nBuOH was
used as coupling partner instead of MeOH. UPLC−MS (ESI+) m/z
1
422.17 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.22 (br s, 1H),
Ethyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (34). Com-
pound 34 was synthesized following the same procedure as the one
outlined for compound 33, the only difference being that EtOH was
used as coupling partner instead of MeOH. UPLC−MS (ESI+) m/z
8.14 (dd, J = 1.6, 8.1 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.40 (d, J =
8.1 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 5.52 (s, 2H), 4.05 (t, J = 6.6
Hz, 2H), 4.04 (s, 3H), 1.60−1.53 (m, 2H), 1.53−1.45 (m, 4H),
1.27−1.19 (m, 2H), 0.78 (t, J = 7.4 Hz, 3H).
Cyclobutyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (42).
Compound 42 was synthesized following the same procedure as the
one outlined for compound 33, the only difference being that
cyclobutanol was used as coupling partner instead of MeOH. UPLC−
1
394.14 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.23 (br s, 1H),
8.14 (dd, J = 1.4, 8.1 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.41 (d, J =
8.1 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 5.52 (s, 2H), 4.11 (q, J = 7.1
Hz, 2H), 4.04 (s, 3H), 1.60−1.53 (m, 2H), 1.53−1.44 (m, 2H), 1.14
(t, J = 7.0 Hz, 3H).
1
MS (ESI+) m/z 420.16 (MH+). H NMR (600 MHz, DMSO-d6) δ
Propyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (35). Com-
pound 35 was synthesized following the same procedure as the one
outlined for compound 33, the only difference being that nPrOH was
used as coupling partner instead of MeOH. UPLC−MS (ESI+) m/z
8.23 (br s, 1H), 8.15 (dd, J = 1.5, 8.0 Hz, 1H), 8.00 (d, J = 8.1 Hz,
1H), 7.41 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 5.52 (s, 2H),
4.98−4.89 (m, 1H), 4.04 (s, 3H), 2.28−2.20 (m, 2H), 1.98−1.89 (m,
2H), 1.74−1.66 (m, 1H), 1.61−1.52 (m, 3H), 1.52−1.47 (m, 2H).
Cyclopentyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (43).
Compound 43 was synthesized following the same procedure as the
one outlined for compound 33, the only difference being that
cyclopentanol was used as coupling partner instead of MeOH.
UPLC−MS (ESI+) m/z 434.17 (MH+). 1H NMR (600 MHz,
DMSO-d6) δ 8.22 (br s, 1H), 8.13 (dd, J = 1.5, 8.0 Hz, 1H), 7.99 (d, J
= 8.0 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 5.51
(s, 2H), 5.10 (tt, J = 2.7, 5.9 Hz, 1H), 4.04 (s, 3H), 1.83−1.72 (m,
2H), 1.59−1.52 (m, 4H), 1.52−1.43 (m, 6H).
1
408.16 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.25−8.21 (br s,
1H), 8.14 (dd, J = 1.5, 8.1 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.40 (d,
J = 8.1 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 5.51 (s, 2H), 4.04 (s, 3H),
4.01 (t, J = 6.6 Hz, 2H), 1.59−1.55 (m, 2H), 1.55−1.47 (m, 4H),
0.79 (t, J = 7.5 Hz, 3H).
Isopropyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (36). Com-
pound 36 was synthesized following the same procedure as the one
i
outlined for compound 33, the only difference being that PrOH was
used as coupling partner instead of MeOH. UPLC−MS (ESI+) m/z
Cyclohexyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (44).
Compound 44 was synthesized following the same procedure as the
one outlined for compound 33, the only difference being that
cyclohexanol was used as coupling partner instead of MeOH. UPLC−
1
408.16 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.23 (br s, 1H),
8.15 (dd, J = 1.5, 8.1 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.41 (d, J =
8.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 5.51 (s, 2H), 4.98−4.90 (m,
1H), 4.04 (s, 3H), 1.58−1.51 (m, 2H), 1.51−1.45 (m, 2H), 1.16 (d, J
= 6.2 Hz, 6H).
1
MS (ESI+) m/z 448.19 (MH+). H NMR (600 MHz, DMSO-d6) δ
tert-Butyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]-
triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (37). See the
preparation of compound 4 described in WO 2013/092739. UPLC−
8.23 (br s, 1H), 8.14 (dd, J = 1.4, 8.1 Hz, 1H), 7.99 (d, J = 8.0 Hz,
1H), 7.41 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 5.51 (s, 2H),
4.73 (tt, J = 3.7, 8.1 Hz, 1H), 4.04 (s, 3H), 1.75−1.66 (m, 2H), 1.60−
1.51 (m, 4H), 1.51−1.46 (m, 2H), 1.43−1.32 (m, 3H), 1.31−1.24
(m, 2H), 1.21−1.12 (m, 1H).
Cycloheptyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (45).
Compound 45 was synthesized following the same procedure as the
one outlined for compound 33, the only difference being that
cycloheptanol was used as coupling partner instead of MeOH.
UPLC−MS (ESI+) m/z 462.20 (MH+). 1H NMR (600 MHz,
DMSO-d6) δ 8.22 (br s, 1H), 8.14 (dd, J = 1.5, 8.1 Hz, 1H), 7.99 (d, J
= 7.8 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 5.51
(s, 2H), 4.89 (tt, J = 4.1, 8.0 Hz, 1H), 4.04 (s, 3H), 1.81−1.72 (m,
2H), 1.62−1.52 (m, 4H), 1.52−1.30 (m, 10H).
1
MS (ESI+) m/z 422.17 (MH+). H NMR (300 MHz, DMSO-d6) δ
8.25 (br s, 1H), 8.16 (dd, J = 1.5, 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz,
1H), 7.40 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 5.51 (s, 2H),
4.04 (s, 3H), 1.54−1.47 (m, 2H), 1.45−1.40 (m, 2H), 1.38 (s, 9H).
[(1R)-1-Methylpropyl] 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-
5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate
(38). See the preparation of compound 2 described in WO 2013/
092739. UPLC−MS (ESI+) m/z 422.17 (MH+). 1H NMR (600
MHz, DMSO-d6) δ 8.24−8.20 (br s, 1H), 8.14 (dd, J = 1.4, 8.0 Hz,
1H), 7.99 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.23 (d, J =
8.2 Hz, 1H), 5.51 (s, 2H), 4.85−4.71 (m, 1H), 4.04 (s, 3H), 1.60−
1.56 (m, 1H), 1.56−1.50 (m, 2H), 1.50−1.41 (m, 3H), 1.13 (d, J =
6.3 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H).
Oxetan-3-yl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (46).
Compound 46 was synthesized following the same procedure as the
one outlined for compound 33, the only difference being that 3-
[(1S)-1-Methylpropyl] 1-[8-methoxy-5-(1-oxo-3H-isobenzofuran-
5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate
(39). See the preparation of compound 1 described in WO 2013/
092739. UPLC−MS (ESI+) m/z 422.17 (MH+). 1H NMR (600
P
https://dx.doi.org/10.1021/acs.jmedchem.0c00797
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Drug Annotation
oxetanol was used as coupling partner instead of MeOH. UPLC−MS
7.40 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 5.51 (s, 2H), 4.51
(t, J = 5.3 Hz, 1H), 4.10−3.97 (m, 4H), 3.92 (dd, J = 6.2, 10.7 Hz,
1H), 3.20 (t, J = 5.5 Hz, 2H), 1.87−1.68 (m, J = 6.6 Hz, 1H), 1.62−
1.54 (m, 2H), 1.54−1.46 (m, 2H), 0.75 (d, J = 6.9 Hz, 3H).
1
(ESI+) m/z 422.14 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.25
(br s, 1H), 8.16 (dd, J = 1.3, 8.1 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H),
7.42 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 5.51 (s, 2H), 5.42
(t, J = 5.8 Hz, 1H), 4.81−4.76 (m, 2H), 4.44 (dd, J = 4.9, 7.9 Hz,
2H), 4.04 (s, 3H), 1.69−1.61 (m, 2H), 1.61−1.51 (m, 2H).
[(3R)-Tetrahydrofuran-3-yl] 1-[8-methoxy-5-(1-oxo-3H-isoben-
zofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropane-
carboxylate (47). Compound 47 was synthesized following the same
procedure as the one outlined for compound 33, the only difference
being that (3R)-tetrahydrofuran-3-ol was used as coupling partner
instead of MeOH. UPLC−MS (ESI+) m/z 436.15 (MH+). 1H NMR
(600 MHz, DMSO-d6) δ 8.23 (br s, 1H), 8.14 (dd, J = 1.4, 8.1 Hz,
1H), 8.00 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.24 (d, J =
8.3 Hz, 1H), 5.52 (s, 2H), 5.31−5.27 (m, 1H), 4.04 (s, 3H), 3.77 (dd,
J = 4.6, 10.4 Hz, 1H), 3.68−3.60 (m, 3H), 2.11 (dtd, J = 6.3, 8.2, 13.6
Hz, 1H), 1.87−1.79 (m, 1H), 1.62−1.55 (m, 2H), 1.54−1.47 (m,
2H).
[(3S)-Tetrahydrofuran-3-yl] 1-[8-methoxy-5-(1-oxo-3H-isoben-
zofuran-5-yl)-[1,2,4]triazolo-[1,5-a]pyridin-2-yl]cyclopropane-
carboxylate (48). Compound 48 was synthesized following the same
procedure as the one outlined for compound 33, the only difference
being that (3S)-tetrahydrofuran-3-ol was used as coupling partner
instead of MeOH. UPLC−MS (ESI+) m/z 436.15 (MH+). 1H NMR
(600 MHz, DMSO-d6) δ 8.23 (br s, 1H), 8.14 (dd, J = 1.5, 8.0 Hz,
1H), 8.00 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.24 (d, J =
8.3 Hz, 1H), 5.51 (s, 2H), 5.32−5.26 (m, 1H), 4.04 (s, 3H), 3.78 (dd,
J = 4.6, 10.4 Hz, 1H), 3.69−3.60 (m, 3H), 2.11 (dtd, J = 6.3, 8.2, 13.5
Hz, 1H), 1.87−1.79 (m, 1H), 1.62−1.55 (m, 2H), 1.55−1.47 (m,
2H).
Tetrahydropyran-4-yl 1-[8-methoxy-5-(1-oxo-3H-isobenzofuran-
5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate
(49). Compound 49 was synthesized following the same procedure as
the one outlined for compound 33, the only difference being that
tetrahydropyran-4-ol was used as coupling partner instead of MeOH.
UPLC−MS (ESI+) m/z 450.17 (MH+). 1H NMR (600 MHz,
DMSO-d6) δ 8.23 (br s, 1H), 8.13 (dd, J = 1.4, 8.1 Hz, 1H), 8.00 (d, J
= 8.1 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 5.51
(s, 2H), 4.93 (tt, J = 3.8, 7.8 Hz, 1H), 4.04 (s, 3H), 3.64 (ddd, J = 3.8,
6.1, 10.7 Hz, 2H), 3.41 (ddd, J = 3.3, 8.0, 11.4 Hz, 2H), 1.81 (ddd, J =
3.4, 6.4, 13.5 Hz, 2H), 1.62−1.56 (m, 2H), 1.56−1.44 (m, 4H).
(2-Hydroxy-2-methylpropyl) 1-[8-methoxy-5-(1-oxo-3H-isoben-
zofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropane-
carboxylate (50). Compound 50 was synthesized following a
procedure similar to the one outlined for compound 33, the major
difference being that 2-methylpropane-1,2-diol was used as coupling
partner instead of MeOH. UPLC−MS (ESI+) m/z 438.17 (MH+).
1H NMR (600 MHz, DMSO-d6) δ 8.24 (br s, 1H), 8.13 (dd, J = 1.4,
2-(Hydroxymethyl)-4-[2-(1-isobutoxycarbonylcyclopropyl)-8-me-
thoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoic Acid (53). Aqueous
NaOH (0.10 M, 1.2 mL, 0.12 mmol) was added to a suspension of
compound 40 (50 mg, 0.12 mmol) in dioxane (5.0 mL), and the
reaction mixture was stirred at rt overnight. More NaOH (0.10 M,
0.24 mL, 0.024 mmol) was then added, and after an additional 1.5 h
of stirring at rt, basic prep HPLC purification afforded the title
1
compound. UPLC−MS (ESI+) m/z 440.18 (MH+). H NMR (600
MHz, DMSO-d6) δ 8.19 (br s, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.93
(dd, J = 1.9, 8.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 8.2 Hz,
1H), 4.90 (s, 2H), 4.03 (s, 3H), 3.84 (d, J = 6.5 Hz, 2H), 1.87−1.72
(m, 1H), 1.59−1.53 (m, 2H), 1.53−1.47 (m, 2H), 0.78 (d, J = 6.7 Hz,
6H).
1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]triazolo-
[1,5-a]pyridin-2-yl]cyclopropanecarboxylic Acid (54). See the
“Preparation 1 to 11” described in WO 2013/092739. The synthesis
of compound 54 on kilogram scale is provided in WO 2014/206903.
UPLC−MS (ESI+) m/z 366.11 (MH+). 1H NMR (600 MHz,
DMSO-d6) δ 8.23 (br s, 1H), 8.13 (dd, J = 1.5, 8.1 Hz, 1H), 8.00 (d, J
= 8.1 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 5.51
(s, 2H), 4.04 (s, 3H), 1.56−1.46 (m, 2H), 1.45−1.35 (m, 2H).
4-[2-(1-Carboxycyclopropyl)-8-methoxy-[1,2,4]triazolo[1,5-a]-
pyridin-5-yl]-2-(hydroxymethyl)benzoic Acid (55). The preparation
is described in WO 2014/206903. UPLC−MS (ESI+) m/z 384.12
(MH+). 1H NMR (300 MHz, DMSO-d6) δ 8.21 (br s, 1H), 7.99 (d, J
= 8.2 Hz, 1H), 7.93 (dd, J = 1.8, 8.1 Hz, 1H), 7.31 (d, J = 8.1 Hz,
1H), 7.21 (d, J = 8.3 Hz, 1H), 4.91 (s, 2H), 4.03 (s, 3H), 1.58−1.47
(m, 2H), 1.47−1.38 (m, 2H).
2-(Hydroxymethyl)-4-[2-[1-(2-hydroxy-2-methylpropoxy)-
carbonylcyclopropyl]-8-methoxy-[1,2,4]triazolo[1,5-a]pyridin-5-yl]-
benzoic Acid (72). A suspension of compound 50 (60 mg, 0.14
mmol) and solid K3PO4 (44 mg, 0.21 mmol) in MeCN (10 mL) and
water (4.0 mL) was stirred at rt for 3 days. Acidic prep HPLC
purification afforded the title compound. UPLC−MS (ESI+) m/z
1
456.18 (MH+). H NMR (600 MHz, DMSO-d6) δ 8.13 (br s, 1H),
7.97 (d, J = 8.1 Hz, 1H), 7.92 (dd, J = 2.0, 8.3 Hz, 1H), 7.30 (d, J =
8.1 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 4.86 (s, 2H), 4.55 (s, 1H), 4.03
(s, 3H), 3.82 (s, 2H), 1.65−1.56 (m, 2H), 1.52−1.47 (m, 2H).
PDE4 Assay. Inhibition of PDE4 activity was measured using the
IMAP (immobilized metal ion affinity particles) TR-FRET pro-
gressive binding assay (Molecular Devices). In brief, the human
PDE4D catalytic domain was incubated with titrated test compound
and a mixture of nonlabeled cAMP and fluorescein amidite (FAM)
conjugated cAMP. Following a brief incubation, the enzymatic
reaction was stopped by the addition of nanoparticles containing a Tb
labeled TR-FRET donor and capable of binding AMP phospho
groups. Time-resolved FRET was measured on an Envision detection
system and was inversely proportional to PDE4 inhibition.
TNF-α Release. Human peripheral blood mononuclear cells
(PBMCs) were isolated from buffy coats using Lymphoprep tubes
(Medinor AB). The PBMCs were resuspended in RPMI1640 and
preincubated for 30 min with titrated test compounds in 384-well
assay plates at 5000 cells/well. Following 18 h stimulation with 10 ng/
mL lipopolysaccharide (Sigma), secreted TNF-α was quantified by
AlphaLISA (PerkinElmer).
Modeling. Docking of 3 in the enol tautomeric form to PDE4 was
performed using the crystal structure of PDE4 complexed roflumilast
PDB code 1XOQ). 3 was docked flexibly with Glide XP with 0.8
scaling of the protein atoms and default settings for ligand scaling.
The highest scoring docking pose is shown in Figure 2.
A five-point pharmacophore model based on the docked
conformation of 3 was generated with Phase. Low-energy
conformations of 13 were generated with Macromodel (OPLS3e,
water, TNCG minimization) and fitted to the pharmacophore
hypothesis. The best fit (shown in Figure 4) is obtained with a
conformation with a relative energy of 0.142 kJ/mol.
8.1 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.24
(d, J = 8.2 Hz, 1H), 5.51 (s, 2H), 4.54 (s, 1H), 4.04 (s, 3H), 3.83 (s,
2H), 1.66−1.56 (m, 2H), 1.56−1.47 (m, 2H), 0.98 (s, 6H).
[(2R)-3-Hydroxy-2-methylpropyl] 1-[8-Methoxy-5-(1-oxo-3H-iso-
benzofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropane-
carboxylate (51). A mixture of compound 54 (100 mg, 0.274 mmol),
(2R)-3-bromo-2-methylpropan-1-ol (200 mg, 1.31 mmol), and
Cs2CO3 (44,6 mg, 0.137 mmol) in DMSO (4.0 mL) was stirred
over the weekend. Acidic prep HPLC purification afforded the title
1
compound. UPLC−MS (ESI+) m/z 438.17 (MH+). H NMR (300
MHz, CDCl3) δ 8.11 (br s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.98 (dd, J
= 1.4, 8.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H),
5.43 (s, 2H), 4.23 (dd, J = 4.6, 11.0 Hz, 1H), 4.10 (s, 3H), 4.01 (dd, J
= 7.5, 11.0 Hz, 1H), 3.53−3.34 (m, 2H), 2.00−1.90 (m, 1H), 1.76−
1.68 (m, 2H), 1.67−1.59 (m, 2H), 0.85 (d, J = 6.9 Hz, 3H).
[(2S)-3-Hydroxy-2-methylpropyl] 1-[8-Methoxy-5-(1-oxo-3H-iso-
benzofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropane-
carboxylate (52). Compound 52 was synthesized following the same
procedure as the one outlined for compound 51, the only difference
being that (2S)-3-bromo-2-methylpropan-1-ol was used as alkylation
reagent instead of (2R)-3-bromo-2-methylpropan-1-ol. UPLC−MS
1
(ESI+) m/z 438.17 (MH+). H NMR (300 MHz, DMSO-d6) δ 8.22
(br s, 1H), 8.13 (dd, J = 1.5, 8.1 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H),
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c00797
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
MacroModel, Phase, Glide, and Maestro were used as implemented
specific LC−MS/MS methods using an API 5500 QTRAP mass
spectrometer.
The pharmacokinetic profile of 40 in minipig (2 female, 2 male,
̈
̈
in the Schrodinger software suite; Schrodinger, LLC, New York, NY.
Crystallography. The crystal structure of 40 complexed with
PDE4D was solved to a resolution of 1.6 Å at SARomics Biostructures
AB, Lund, Sweden. Hydrogens were added, H-bonds were optimized,
and the complex was minimized using the Protein Preparation Wizard
implemented in Maestro.
̈
Gottingen) and monkey (2 female, 2 male cynomolgus) was
determined in a 24 h infusion study dosed at 0.5 and 2.0 mg/kg
(0.1 and 0.4 mg/mL at 5 mL/kg) in 100% tetraethylene glycol, and
blood samples were taken at 0.25, 0.5, 1, 3, 7, and 24 h after the start
of infusion and 6, 12, and 24 h after the end of the 24 h infusion
period. Blood was sampled in tubes EDTA-K3 as anticoagulant,
centrifuged to prepare plasma, and stabilized with 50 mg of ascorbic
acid prior to storage at −80 °C until analysis of 40, 53, 54, 55, 50, 71
by compound specific LC−MS/MS methods using an API 5500
QTRAP mass spectrometer. Pharmacokinetic parameters were
derived from a noncompartment model in Phoenix64 (Pharsight,
Certara).
HLM Clearance. Metabolic stability in human liver microsomes
was measured as compound clearance following a 40 min incubation
with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) at
a test compound concentration of 0.5 μM. Samples are taken out
before addition of NADPH and at 5, 10, 20, and 40 min thereafter,
and protein precipitation using cold acetonitrile with added internal
standard is performed prior to centrifugation and sample analysis
using compound specific LC−MS/MS methods. Control incubations
in buffer and without NADPH are performed in the same manner.
Compound depletion is determined and apparent clearance
calculated.
Hepatocyte Clearance. Metabolic stability in cryopreserved
human, rat, and minipig hepatocytes is determined with 1 million
cells/mL suspended in buffer at a test compound concentration of 0.5
μM in duplicate. After a preheating period of 10 min the test
compound is added and the incubation continues with stirring for 0,
5, 10, 30, and 60 min. This is followed by protein precipitation using
cold acetonitrile with added internal standard, centrifugation, and
sample analysis using compound specific LC−MS/MS methods.
Control incubations in buffer are performed in the same manner. The
compound depletion over time was used to estimate the elimination
rate constant, from which the apparent intrinsic clearance (CLapp) and
half-life (t1/2) were calculated. The hepatic clearance is calculated
using the well-stirred model.29
Human Keratinocyte Stability. The half-life of test compounds
is determined in a human keratinocyte stability assay as surrogate for
stability in the human skin. Plated human epidermal keratinocytes (1
× 104 cells/well) are incubated for up to 24 h with a test compound
concentration of 1 μM in duplicate. Samples are taken at 0, 1, 2, 4,
and 24 h and followed by protein precipitation using cold acetonitrile
with added internal standard, centrifugation, and sample analysis
using compound specific LC−MS/MS methods. Control incubations
in growing medium are performed in the same manner. Compound
half-life is reported.
Dermal Pharmacokinetics. The dermal concentration of 40, 53,
̈
54, and 55 was investigated in female Gottingen minipigs after single
topical application of 40 in 2.5 and 1 mg/g monoglyceride
formulations (10 μL/cm2 on a 4 cm2 area on the belly/flank area).
After dosing, excess formulation was removed, and after 24 h the test
sites were tape stripped 2 times in order to remove surplus test item
from stratum corneum followed by 10 times to remove stratum
corneum. Punch biopsies of 8 mm were taken from each of the tape-
stripped application sites and the biopsies were snap frozen in liquid
nitrogen until analysis. The skin biopsies were homogenized, and
protein precipitation was performed with acetonitrile containing
internal standard and centrifugation prior to analysis until analysis of
the samples by compound specific LC−MS/MS methods using an
API 5500 QTRAP mass spectrometer.
In Vivo Study Procedures. Animal experimental procedures as
well as animal housing and husbandry were in compliance with
Danish legislation, EU Directive, as well as the project license
approved by the Danish authorities for animal experimentation.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00797.
■
sı
HPLC traces of key compounds (PDF)
Molecular formula strings and some data (CSV)
Whole Blood Stability. Compounds were incubated in diluted
fresh rat, dog, cynomolgus monkey, or human whole blood (heparin
stabilized, 50% blood and 50% phosphate buffer with 1% bovine
serum albumin) for 120 min at 0.1 μM at 37 °C, and the half-lives
were determined. Samples were taken at 0, 15, 30, 60, and 120 min,
and reactions were terminated by addition of acetonitrile containing
analytical internal standard (IS). All compounds were tested in
duplicate. Testing for compound depletion used compound specific
LC−MS/MS methods. The elimination rate constants (k) were
Accession Codes
The atomic coordinates of 40 (LEO 39652) bound to PDE4D
has been deposited with the Protein Data Bank under the
accession code 6ZBA.
AUTHOR INFORMATION
Corresponding Author
■
calculated from peak areas, and the half-lives were determined (t1/2
ln 2/k).
=
Simon F. Nielsen − Drug Design, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark;
orcid.org/0000-0001-8263-4469; Phone: 0045 2551
8760; Email: sfndk@leo-pharma.com
Metabolite Identification. A cross-species comparison of
metabolite profiles for both 40 and 53 was performed in hepatocytes
(2 h, 10 μM incubation) and whole blood (2 h, 10 μM incubation)
from human, rat, minipig, and monkey. The metabolite profiles were
from using UPLC/TOF-MS analysis of the samples and with both
targeted and untargeted search for metabolites using Metabolynx
software (Waters) followed by confirmation with analytical standards
of metabolites of 53, 54, 55, 50 and 72.
In Vivo Pharmacokinetics. The pharmacokinetic profile of 40
was determined in rat (n = 3, male, Sprague Dawley) after an
intravenous (iv) injection in 20% cyclodextrin once at 0.075 mg/kg
(50 μg/mL, 1.5 mL/kg). Blood is collected after 1, 3, 6, 9, 12, 15, and
30 min from the tongue vein in lithium-heparin vacutainers and
immediately hereafter, transferred to Micronic vials containing
heparin water, ratio blood−water (1:1). Protein precipitation with
ice-cold acetonitrile (containing internal standard for bioanalysis) is
performed, and samples were kept on ice until centrifugation and
stored at −20 °C until analysis of 40, 53, 54, and 55 by compound
Authors
Jens Larsen − Drug Design, LEO Pharma Global Research &
Development, 2750 Ballerup, Denmark
Maja Lambert − Drug Design, LEO Pharma Global Research &
Development, 2750 Ballerup, Denmark
Henrik Pettersson − Drug Design, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
Thomas Vifian − Drug Design, LEO Pharma Global Research
& Development, 2750 Ballerup, Denmark
Mogens Larsen − Drug Design, LEO Pharma Global Research
& Development, 2750 Ballerup, Denmark
Anna Ollerstam − Skin Research, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
R
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(6) Halling-Overgaard, A. S.; Kezic, S.; Jakasa, I.; Engebretsen, K. A.;
Maibach, H.; Thyssen, J. P. Skin absorption through atopic dermatitis
skin: a systematic review. Br. J. Dermatol. 2017, 177, 84−103.
(7) Moustafa, F.; Feldman, S. R. A review of phosphodiesterase-
inhibition and the potential role for phosphodiesterase 4-inhibitors in
clinical dermatology. Dermatol. Online J. 2014, 20, 22608.
(8) Bodor, N.; Buchwald, P. Soft drug design: general principles and
recent applications. Med. Res. Rev. 2000, 20 (1), 58−101.
(9) Felding, J.; Sørensen, M. D.; Poulsen, T. D.; Larsen, J.;
Andersson, C.; Refer, P.; Engell, K.; Ladefoged, L. G.; Thormann, T.;
Vinggaard, A. M.; Hegardt, P.; Søhoel, A.; Nielsen, S. F. Discovery
and early clinical development of 2-{6-[2-(3,5-dichloro-4-pyridyl)-
acetyl]-2,3-dimethoxyphenoxy}-N-propylacetamide (LEO 29102), a
soft-drug inhibitor of phosphodiesterase 4 for topical treatment of
atopic dermatitis. J. Med. Chem. 2014, 57 (14), 5893−5903.
(10) Aprile, S.; Serafini, M.; Pirali, T. Soft drugs for dermatological
applications: recent trends. Drug Discovery Today 2019, 24 (12),
2234−2246.
Pontus Hegardt − Global Clinical Operations Projects, LEO
Pharma Global Research & Development, 2750 Ballerup,
Denmark
Cecilia Eskilsson − Preclinical Development, LEO Pharma
Global Research & Development, 2750 Ballerup, Denmark
Steen Laursen − Preclinical Development, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
Anders Soehoel − Clinical Pharmacology, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
Tine Skak-Nielsen − Skin Research, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
Lene M. Hansen − Early Formulation and Analytics, LEO
Pharma Global Research & Development, 2750 Ballerup,
Denmark
Nina Ø. Knudsen − Early Formulation and Analytics, LEO
Pharma Global Research & Development, 2750 Ballerup,
Denmark
Stefan Eirefelt − Drug Design, LEO Pharma Global Research &
Development, 2750 Ballerup, Denmark
(11) LEO 29102 Cream in the Treatment of Atopic Dermatitis.
https://clinicaltrials.gov/ct2/show/NCT01037881 (accessed May 1,
2020); https://www.clinicaltrialsregister.eu/ctr-search/rest/
download/result/attachment/2009-013792-22/1/17100 (accessed
May 1, 2020)
Morten D. Sørensen − Drug Design, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
Tatiana G. Stilou − Skin Research, LEO Pharma Global
Research & Development, 2750 Ballerup, Denmark
(12) Fukami, T.; Yokoi, T. The emerging role of human esterases.
Drug Metab. Pharmacokinet. 2012, 27 (5), 466−477.
(13) Berry, L. M.; Wollenberg, L.; Zhao, Z. Esterase activities in the
blood, liver and intestine of several preclinical species and humans.
Drug Metab. Lett. 2009, 3 (2), 70−77.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00797
(14) Oesch, F.; Fabian, E.; Oesch-Bartlomowicz, B.; Werner, C.;
Landsiedel, R. Drug-metabolizing enzymes in the skin of man, rat, and
pig. Drug Metab. Rev. 2007, 39 (4), 659−698.
Notes
The authors declare no competing financial interest.
(15) Felding, J.; Sørensen, M. D. Triazolopyridines as Phosphodies-
terase Inhibitors for treatment of Dermal Diseases. PTC Int. Appl.
WO2008/125111 A1, Oct 23, 2008.
ACKNOWLEDGMENTS
■
Saromics Biostructures AB is gratefully acknowledge for expert
assistance in determination of the structure of the PDE4-40
complex.
(16) Hatzelmann, A.; Schudt, C. Anti-inflammatory and immuno-
modulatory potential of the novel PDE4 inhibitor roflumilast in vitro.
J. Pharmacol. Exp. Ther. 2001, 297 (1), 267−279.
(17) McGrath, J. A.; Eady, R. A. J.; Pope, F. M. Anatomy and
Organization of Human Skin. In Rook’s Textbook of Dermatology, 7th
ed.; Burns, T., Breathnach, S., Cox, N., Griffiths, C., Eds.; Blackwell
Publishing: Oxford, U.K., 2004; pp 4190.
ABBREVIATIONS USED
■
AD, atopic dermatitis; CES, carboxylesterase; Hep, hepatoto-
cytes; HLM, human liver microsome; KC, keratinocyte;
PBMC, human peripheral blood mononuclear cell; PON,
paraoxonase; Th1, T helper cell type 1; Th2, T helper cell type
2; Th17, T helper cell type 17; TCI, topical calcineurin
inhibitor; TCS, topical corticosteroid; WB, whole blood
(18) Man, M. Q.; Hatano, Y.; Lee, S. H.; Man, M.; Chang, S.;
Feingold, K. R.; Leung, D. Y.; Holleran, W.; Uchida, Y.; Elias, P. M.
Characterization of a hapten-induced, murine model with multiple
features of atopic dermatitis: structural, immunological, and
biochemical changes following single versus multiple oxazolone
challenges. J. Invest. Dermatol. 2008, 128, 79−86.
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