Efficacious inhaled PDE4 inhibitors with low emetic potential and long duration of action for the treatment of COPD

Article abstract of DOI:10.1021/jm5001216

Oral phosphodiesterase 4 (PDE4) inhibitors, such as cilomilast and roflumilast, have been shown to be efficacious against chronic obstructive pulmonary disease (COPD). However, these drugs have been hampered by mechanism-related side effects such as nause

Full text of DOI:10.1021/jm5001216

Article
pubs.acs.org/jmc
Efficacious Inhaled PDE4 Inhibitors with Low Emetic Potential and
Long Duration of Action for the Treatment of COPD
Chris De Savi,*,†,‡ Rhona J. Cox,*,†,§ Daniel J. Warner,† Anthony R. Cook,† Mark R. Dickinson,†
Amy McDonough,†,∥ Louis C. Morrill,† Beth Parker,† Glen Andrews,† Simon S. Young,†,∥
Peter S. Gilmour,†,∥ Rob Riley,†,∥ and Matthew S. Dearman†,§
†AstraZeneca R&D Charnwood, Loughborough, Leicestershire, LE11 5RH, U.K.
‡Oncology iMed, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
§Respiratory, Inflammation & Autoimmunity iMed, AstraZeneca R&D Molndal, Pepparedsleden, 431 83 Molndal, Sweden
̈
̈
∥AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, U.K.
S
* Supporting Information
ABSTRACT: Oral phosphodiesterase 4 (PDE4) inhibitors,
such as cilomilast and roflumilast, have been shown to be
efficacious against chronic obstructive pulmonary disease
(COPD). However, these drugs have been hampered by
mechanism-related side effects such as nausea and emesis at
high doses. Compounds administered by inhalation are
delivered directly to the site of action and may improve the
therapeutic index required to overcome side effects. This paper
describes systematic and rational lead optimization to deliver
highly potent, long-acting, and efficacious preclinical inhaled
PDE4 inhibitors with low emetic potential.
shown clinical efficacy, and roflumilast has a superior therapeutic
index over emesis. However, nausea and vomiting still limit the
oral dose of PDE4 inhibitors (Figure 1).
INTRODUCTION
■
Chronic obstructive pulmonary disease (COPD) is charac-
terized by persistent airflow limitation that is usually
progressive and is associated with an enhanced chronic
inflammatory response in the airways and lungs to noxious
particles and gases.1 It is a major public health burden, with a
global prevalence of 10.1%,2 causing 3 million deaths worldwide
in 20113 and predicted to be the fourth leading cause of death
by 2030.4 The economic burden associated with the disease is
substantial, with the total costs of COPD in the U.S. estimated
at $49.8 billion in 2010.5 COPD-associated inflammation
responds only weakly to corticosteroids,6 and new treatments
are urgently required.
Phosphodiesterase 4 (PDE4) hydrolyzes the phosphodiester
bond of cAMP to produce inactive AMP. Inhibitors of PDE4
elevate intracellular cAMP concentration, which leads to
activation of specific protein phosphorylation cascades. These
in turn reduce a wide variety of inflammatory mediators and
inflammatory cell functions.7 PDE4 inhibitors reduce pulmonary
inflammation and improve lung function in preclinical models of
asthma and COPD, and several PDE4 inhibitors have advanced
into clinical trials for the treatment of COPD and other
inflammatory diseases.8
Most clinical investigations have focused on oral delivery
of PDE4 inhibitors. The first-generation inhibitor R-rolipram
(1) caused emesis, a mechanistic side-effect of PDE4 inhibitors,
at effective anti-inflammatory doses.9 More recently, second-
generation inhibitors cilomilast (2)10 and roflumilast (3)11 have
Figure 1. Representative oral and inhaled PDE4 inhibitors.
Received: January 23, 2014
© XXXX American Chemical Society
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Drugs to treat respiratory diseases can be inhaled, which
reduces systemic exposure and associated side effects. Pfizer and
GlaxoSmithKline have both reported inhaled PDE4 inhibitors
which have progressed to the clinic. Others have also recently
reported the design and discovery of novel inhaled PDE4
inhibitors.12,13 Pfizer’s UK-500,001 (4) showed preclinical
efficacy in a variety of species but has since been discontinued
because of lack of efficacy in patients suffering from moderate to
severe COPD.14 Although the drug was generally well tolerated,
mechanism-related side effects were observed in the higher dose
groups. GSK256066 (5) was in clinical development for allergic
rhinitis, asthma, and COPD but is not currently listed in the
company’s pipeline. It is very potent in vitro and has
demonstrated potent and long-lasting anti-inflammatory effects
in animal models of pulmonary inflammation.15 Furthermore,
no emesis has been reported in a ferret model.16 Clinically,
5 significantly reduced early and late stage asthmatic response to
allergen challenge in mild asthmatics after 1 week of treatment.17
activity seen for 4 in our in-house rat lung inflammation models
could be due to its moderate potency. High total lung
concentrations were not able to compensate for this potency in
these experiments, potentially as a consequence of low solubility.
In our model, 5 had a variable effect on lung neutrophilia
with no clear dose response. It inhibited lung neutrophilia at
0.03 mg/mL equivalent nebulizer concentration, resulting in a
measured terminal lung concentration of 38 nM. No significant
effects were observed at 0.01 or 0.1 mg/mL (Figure S2A,
Supporting Information). No doses of 5 resulted in significant
inhibition of lung inflammation when dosed 12 h before the
inflammatory challenge despite the highest dose achieving total
lung concentrations of up to 356 nM (Figure S2B, Supporting
Information). The reason for the variable efficacy of 5 is not
clear; the lack of a relationship between compound level and
biological effect is surprising. We have hypothesized that the lack
of significant activity of 5 at 12 h could be due to an inconsistent
availability of free compound in the biophase and activity
associated with poorly soluble compound in the lung,19 similar to
4. However, we cannot discount that using a different
formulation or in vivo model may lead to different results.
Studies by GSK on LPS-induced lung inflammation in rats
showed that high levels of 5 were seen in lung tissue up to 40 h,
when the inflammatory effect had declined.16 GSK has suggested
that 5 is retained in lung tissue because of the lipophilic nature of
this compound, but there is little relationship between lung
matrix levels of 5 and duration of anti-inflammatory effect. It is
likely that high levels of 5 are being retained in the lung but that
the majority of compound is either in solid form and/or in lipid
compartments in the lung and the levels of free compound in the
intracellular cytoplasm (the site of PDE4) are too low to exert a
pharmacological effect.
Design. Our strategy was also to reduce mechanism-based
side effects by limiting systemic exposure, but the overall
preclinical profile of our compounds differed. We aimed to
find compounds with high whole blood potency to give a low
(<15 μg/kg) predicted dose to man, along with high solubility
(>50 μM) and >8 h half-life in rat in vivo iv PK, which correlates
well with i.t. PK.20 Low free fraction in human plasma (<5%) and
low preclinical bioavailability should limit systemic exposure and
reduce emetic potential. While there are some reports suggesting
that inhibition of subtype PDE4B leads to anti-inflammatory
effects and that inhibition of PDE4D causes emesis,21 we felt that
it would be difficult to achieve sufficiently high margins using this
approach. Additionally, we required appropriate solid state
properties for inhalation, i.e., high crystallinity, low hygro-
scopicity, no low temperature thermal events, and micronizable
for use in a dry powder inhaler. Together with in vivo activity in
both LPS dose response and duration experiments, we believed
this profile would provide confidence in a once daily predicted
inhaled dose in the clinic.
RESULTS AND DISCUSSION
■
In Vitro and in Vivo Data for 4 and 5. The medicinal
chemistry strategy adopted by GSK was to deliver an exquisitely
potent PDE4 compound with low aqueous solubility and oral
bioavailability.18 The Pfizer strategy appeared to focus on both
high metabolic clearance and high plasma protein binding to
minimize systemic exposure (Table 1).
Table 1. AstraZeneca Data Generated on 4 and 5
property
4
5
a
human PDE4B enzyme pIC50
9.2 (6)
8.4 (10)
NT
>11 (2)
a
PBMC TNF-α pIC50
11.5 (7)
a
human whole blood TNF-α pIC50
9.5 (9)
a
rat whole blood TNF-α pIC50
7.4 (2)
9.6 (2)
a
PPB %free human/rat
0.17/1.7 (1)
<1 (1)
1.5/2.4 (1)
1.2 (1)
a
aqueous solubility pH 7.4 (μM)
a
MW/clogP/log D
500/5.3/4.4 (1)
116 (1)
519/3.8/2.7 (1)
>150 (1)
human microsomal Clint
a
(μL/min/mg)
rat hepatocyte Clint
76 (1)
130 (1)
a
(μL/min/106 cells)
b
rat pharmacokinetics at 1 mg/kg:
Cl (mL/min/kg)
97
32
V
dss/Vz (L/kg)
22/63
7.6
0.75/40
14.9
8.1
iv t1/2 (h)
i.t. t1/2 (h)
NT
c
in vivo efficacy in rat (inhaled):
LPS DRC; lowest efficacious inactive at all doses 0.03 mg/mL
nebulizer concentration
(LENC)
LPS duration
NT
inactive
a
b
c
Numbers of replicates are stated in brackets. n = 2−3 animals. Data
from a single experiment, n = 8−10 animals per treatment group. NT
= not tested.
We have previously disclosed several series of both neutral and
basic highly potent pyridopyrimidinedione PDE4 inhibitors for
inhaled delivery,22,23 of which 6 and 7 are representative
examples (Figure 2). This paper describes the search for a
structurally differentiated back-up series.24
During the early phases of the project, a significant number of
pyridopyrimidinediones were profiled in our LPS dose response
assays. At the time, there was no method to confidently predict
whether a compound would show efficacy and subsequent
duration of effect, so a statistical analysis was undertaken to
identify potential measurable parameters that could be used to
prioritize compounds for in vivo studies.
In our assays, 4 was rapidly metabolized and had high plasma
protein binding and was therefore likely to achieve very low systemic
exposure. It was inactive in our rat pulmonary inflammation model
when administered as a nebulized aerosol (Figure S1A, Supporting
Information). An increase in the lung-delivered dose of 4 when
administered by dry powder aerosol resulted in, at best, inconsistent
inhibition of LPS-induced neutrophilia (Figure S1B, Supporting
Information) despite significant deposited lung doses (Figure S1C,
Supporting Information). We have hypothesized that the lack of
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dose response experiments were determined from these
estimated LENC values.
The nominal logistic regression approach was also applied to
20 compounds previously prioritized for duration experiments to
determine whether any of the same parameters were capable of
discriminating actives from inactives at the later time point. On
the basis of this analysis (and given that each compound had
previously shown activity in terms of a dose response), we were
able to estimate the probability of a compound exhibiting
duration of action as a function of log D (Figure S3(c),
Supporting Information):
Figure 2. Representative examples of earlier AstraZeneca PDE4
inhibitors.
1
P(duration of activity) =
1 + e(1.77×log D)−4.45
(4)
Initial quantitative analysis of in vivo efficacy used internal
compounds from the earlier lead optimization program,23
supplemented by a diverse set of published PDE4 inhibitors,
comprising 41 compounds in total. Compounds were
qualitatively flagged as active or inactive, according to whether
they had exhibited significant efficacy at any of the concen-
trations screened in single or multiple dose studies. Nominal
logistic regression was used to independently fit nine in vitro
physicochemical and PK parameters and three in vivo PK
parameters to the qualitative activity flags using JMP.25 The two
parameters exhibiting the most significant correlation (pIC50 in
PBMC and t1/2 from rat iv studies, p < 0.001 in both cases) were
then used in combination to estimate the probability of observing
activity in vivo:
The probability of retaining efficacy for a 12 h period drops
sharply as the log D increases from 2 (71%) to 3 (29%). This
allowed us, on the basis of a single in vitro parameter, to prioritize
only those compounds with the greatest chance of demonstrating
12 h duration, and focus efforts on compounds with equivalent or
lower log D than 5 (log D = 2.7, 54% chance of achieving duration).
It was known from earlier work independently conducted by
AstraZeneca and by Pfizer that a bicyclic or pseudobicyclic
fluoropyridine core substituted by a lipophilic aromatic ring and a
cis-1,4-diaminocyclohexane are beneficial for potency.22,23,26
During development of our biaryl series (7), we discovered that
addition of a base to the biaryl can significantly enhance solubility.
Furthermore, detailed profiling of different chemical classes across
a range of inhaled programs has demonstrated that bases, and
dibases in particular, can provide a long duration in the rat lung
following i.t. administration.27 Compound 8a (Figure 3, Table 2)
1
P(activity in DRC) =
50)−(3.83×log10(t1/2))
1 + e15.85−(1.47×pIC
(1)
Substituting the PBMC pIC50 and t1/2 of 5 into the above
equation gives a >99% chance of activity, whereas the equivalent
substitutions for 4 suggest a 47% chance. A graphical
representation of this model is provided (Figure S3(a),
Supporting Information).
For the purposes of quantitative analysis, the in vivo efficacy of
the compounds was represented by the lowest effective nebulizer
concentration (LENC), i.e., the lowest nebulizer concentration
at which significant efficacy was observed, expressed as:
pLENC = −log10(LENC)
(2)
Using linear regression, we screened the same 12 parameters as
above for statistically significant relationships with pLENC for
the subset of 26 active compounds. In contrast to the qualitative
analysis, quantitative analysis revealed just two statistically
significant relationships: the pIC50 values from the enzyme and
cell potency (PBMC) assays (p = 0.005 and p = 0.011,
respectively). As the correlation between these two parameters
themselves is very high, we opted to base our predictions on the
cellular assay alone, allowing us to estimate the LENC according
to the following equation (Figure S3(b), Supporting Informa-
tion):
Figure 3. Structure of lead compound 8a.
Table 2. Properties of Lead Compound 8a
property
8a
human PDE4B pIC50
PBMC TNF-α pIC50
human PPB % free
10.8
10.2
5.6
aqueous solubility pH 7.4 (μM)
log D
250
3.1
CYP pIC50 (4 isoforms)
rat iv PK Cl/Vdss/Vz/t1/2 (h)
in vivo LPS DRC; LENC
in vivo LPS duration; LENC
<6.0 except 3A4 6.3, 2C9 6.6
69/2.6/76/13
predicted pLENC = 0.48 × pIC50(PBMC) − 1.16
(3)
active at 0.1 mg/mL
active at 0.1 mg/mL
This relationship was heavily based on 24 internal compounds,
as just two reference compounds (2 and 5) exhibited quan-
tifiable activity in our in vivo model. Thus, from the initial
analysis, we were able to identify cell potency and half-life as
qualitative markers of efficacious compounds. Within those
predicted to be active, we could then quantitatively estimate the
LENC based on the cellular potency alone. Dose ranges used in
was discovered during our first lead optimization phase and
appeared to be a good start point for a back-up program.
Variation of the amide and replacements for the morpholine
to decrease the plasma free fraction were investigated first (8).
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Figure 4. Variation of solubility and PPB by changing both the amide and base in structure 8. Colors illustrate CYP3A4 activity (pIC50 > 6.5, red;
6.0 < pIC50 < 6.5, orange; pIC50 < 6.0, green). All compounds have PDE4B pIC50 > 9.5. Star shows 8a.
Figure 5. Structures of para-benzylamines.
Table 3. Properties of Key Compounds from para-
Benzylamine Subseries
In raising lipophilicity, CYP inhibition increased and solubility
decreased to unacceptable levels. A correlation between
solubility and plasma-free fraction suggested that there would
be difficulty in achieving both solubility >50 μM and free fraction
<5% in this subseries (Figure 4). A key finding was that potency
was only retained if the ortho-position was substituted with weak
bases of limited size. However, compound 8a and analogues with
a weak base in the ortho-position generally did not show good
efficacy in the LPS DRC model despite a 96% predicted
likelihood of activity. Morpholine 8a did show efficacy (37%
reduction in LPS-induced bronchoalveolar lavaged neutrophils)
at 0.1 mg/mL but at higher concentrations the inhibition was
lost, suggesting a possibility of local irritancy masking the efficacy
readout at higher doses.
In parallel, we explored compounds with a para-benzylamine
on the biaryl and no ortho-substituent (9) (Figure 5). In this case,
CYP inhibition was a less significant problem and we were able
to raise the log D to achieve a low free fraction. Potency in the
PBMC assay was generally lower than with an ortho-benzylamine,
with the highest potencies in this subseries being achieved with
para-piperazines. Addition of two methyl groups to the
piperazine reduced the PPB free fraction without detrimentally
affecting other properties (Figure 5, Table 3).
property
9a
9b
9c
human PDE4B pIC50
PBMC TNF-α pIC50
human PPB (% free)
10.4
9.6
10.6
10.1
4.5
10.2
9.9
2.0
1.2
aqueous solubility
pH 7.4 (μM)
>1400
580
460
log D
3.2
2.7
2.7
CYP pIC50
all <6.0
<6.0 except 3A4 6.4 all <6.0
61/14/28/5.3
active at 0.03 mg/mL NT
(5 isoforms)
rat iv PK
Cl/Vdss/Vz/t1/2 (h)
79/60/80/12 58/13/30/5.8
in vivo LPS
DRC; LENC
active at
1 mg/mL
the matched pairs in Table 4. The presence of an acceptor
(−N) either adjacent to the amide carbonyl or in a fused ring
was important for high potency, although 2-phenolic compounds
are also potent. The effects of the phenol and nitrogen acceptor
are not additive, suggesting that the two features are involved in
the same binding site interaction.
Potency SAR for the amide appears to be broadly parallel
between the ortho- and para-benzylamine series, as illustrated by
Substitution of the biaryl with a benzylamine in the meta-
position generally led to a drop in potency compared to the
D
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Table 4. PDE4B Enzyme pIC50: Representative Examples to Illustrate SAR
corresponding para-benzylamines. As no other obvious advan-
tages were noted, this subseries was not explored further.
Combination of meta- or para-benzylamines with a phenolic
hydroxyl was also investigated to see if this group could be used
to reduce plasma free fraction (Figure 6, Table 5). In the case of
the meta-benzylamines, addition of a para-hydroxyl led to an
increase in potency but no dramatic change in plasma binding.
Installation of the hydroxyl in other sites around the aryl ring
showed significant drops in cell potency. Addition of a meta-
hydroxyl to a para-benzylamine was generally tolerated, but again
there was no plasma binding advantage. In addition, many of the
hydroxylated benzylamines also carried the theoretical possibility
of elimination of the basic group to form a quinone methide,
which was viewed as an undesirable liability.
Our statistical analysis showed that critical properties needed
for both efficacy and duration were high PBMC pIC50 (ideally
>9.5), low log D, and long half-life (ideally >8 h) in rat PK. While
we had achieved the first two, many compounds synthesized thus
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Figure 6. Substitution on the biaryl with hydroxyl groups.
Table 5. Effects of Hydroxylating the Biaryl
We proposed a further subseries of compounds with bases in
both the ortho- and para-positions (12) and speculated that these
compounds should have high potency, low plasma free fraction,
robust PK and efficacy properties, and perhaps also the potential
for good solid-state properties (Figure 10). For example, earlier
work suggested that compounds with an ortho-benzylamine
tended to crystallize easily to give a nonhygroscopic polymorph.
The results of the first set of ortho,para-dibases to be
synthesized showed good activity in both enzyme and cell assays
but lacked sufficiently high plasma protein binding to meet our
criteria for a clinical candidate. We capitalized on the extensive
knowledge that had already been established in terms of enzyme
activity and human PPB in order to increase PPB in the dibasic
series. Of 1680 compounds with measured PDE4 enzyme
potency, 581 matched the fragmentation scheme depicted in
Figure 11 (top left) and could therefore be used in the model
construction. From this scheme we were able to identify three
unique “cores”, 171 “top”, and 185 “bottom” fragments. A Free−
Wilson model28 for potency was built with a training set R2 of
0.93 and RMSE of 0.22, which we were then able to use to predict
the activity of (3 × 171 × 185) = 94905 virtual structures. As we
were particularly interested in increasing the plasma binding of a
specific subseries (Figure 11, center right), we quickly narrowed
this library to just 171 potential compounds for synthesis,
featuring each of the “top” fragments identified above. This
virtual library covered a range of predicted activities from the
Free−Wilson model and was filtered on predicted pIC50 ≥ 10.2.
Plasma protein binding was predicted29 for the 20 virtual
structures that remained upon filtration, and the results were
visualized as shown to the bottom left of Figure 11. Compounds
that had been synthesized prior to that point (in red) occupy the
bottom left of the plot, i.e., they have comparatively low potency
and plasma binding. Conversely, there are a number of points
situated to the top right of the plot which offered potential
avenues for increasing either of these two parameters or even
property
10a (= 9d) 10b
10c
10d
10e
10f
human PDE4B pIC50
PBMC TNF-α pIC50
human PPB (% free)
10.6
10.0
11
10.5 10.2
10.5 10.1
10.7 10.5
10.4 9.4
10.1
8.5
12
18
16
35
9.1
aqueous solubility
pH 7.4 (μM)
1300
890
>1500 970
>1500 470
log D
2.4
2.4
2.1
2.3
2.2 2.8
far in the project had shown insufficiently long half-life for
progression into efficacy models. In vivo clearance of these
compounds was generally at the rate of liver blood flow, so an
increased volume of distribution (Vz) was needed to prolong
half-life. Because the enzyme binding site appeared to be tolerant
of a wide variety of base positions, the chain length in both para
and meta positions was extended to position the base further
from the biaryl unit and increase basicity. We predicted this
would increase Vz. Low PBMC potency was an issue for many
examples, but a number of those compounds with PBMC
pIC50 > 10 progressed to rat iv PK, showing increased Vz and
correspondingly longer half-lives (Figures 7 and 8).
We were also pleased to discover that these extended bases
showed higher plasma binding for equivalent log D when
compared to earlier compounds, which meant they were more
likely to show both <5% free in plasma and 12 h duration
(Figure 9).
Potency, plasma binding, rat iv PK half-life, and measured log
D were used to prioritize compounds for efficacy and duration
studies. Two lead compounds (11a and 11e) (Table 6) were
investigated further and showed suitable margins in in vitro
toxicology assays. The crystallinity of 11a was investigated and
the free base was found to be crystalline though hygroscopic.
Dibasic compounds made earlier in the project had been
shown to have a 12 h duration of action despite having higher
log D than the top limit for duration in the monobase series.
Figure 7. Structures of extended bases.
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Figure 8. Relationship between rat iv PK half-life and Vz, colored by compound class (11). Orange = para, n = 0; yellow = meta, n = 0; black = ortho, n = 0;
red = para, n = 1; purple = meta, n = 1; green = para, n = 2; blue = meta, n = 2.
Table 6. Properties of Advanced Compounds with Extended
Bases
property
11a
11e
human PDE4B pIC50
PBMC TNF-α pIC50
human PPB (% free)
10.7
10.2
4.5
10.4
10.5
4.2
aqueous solubility pH 7.4
(μM)
130
120
log D
2.6
2.4
rat iv PK Cl/Vdss/Vz/t1/2 (h) 68/21/41/7
74/44/59/9
all <6.0
CYP pIC50 (5 isoforms)
in vivo LPS DRC; LENC
all <6.0
0.03 mg/mL
0.1 mg/mL
ED80 (nebulizer
0.05 mg/mL/60 nM/0.6 μg/kg ND
concentration/lung
concentration/lung
deposited dose)
in vivo LPS duration; LENC active at 0.1 mg/mL
active at 0.1
mg/mL
Figure 9. Variation of plasma binding with log D colored by compound
class as in Figure 8.
both. The compounds shown in blue were selected for synthesis
(Figure 11, bottom right) and this final set of ortho,para-dibasic
compounds was prepared.
Chemistry. All compounds described in this paper were
synthesized from a common intermediate 13 which was made by
amide coupling of chloronicotinic acid 14 to Boc-protected
amine 1526 using propylphosphonic anhydride (T3P) followed
by displacement of chloride by 3-iodophenol (Scheme 1).
The first compound in the series, 8a, was prepared by
deprotection of 13 and amidation, followed by Suzuki coupling
of aryl boronic acid 16 (Scheme 2).
For later compounds, we developed a flexible and high yielding
synthetic strategy which allowed parallel investigation of either
end of the molecule; 13 could either be deprotected and
amidated, followed by Suzuki coupling then functionalization
with a variety of amines (route I) or Suzuki coupling and amine
Figure 10. Structure of ortho,para-dibases.
formation carried out first, followed by amidation with a variety
of acids (route II).
Routes I and II were generally comparable in terms of ease and
overall yield, as illustrated by the synthesis of para-benzylamines
for which both routes were used (Scheme 3).
Sometimes it was more efficient to convert the aryl iodide to
the corresponding boronic ester if the second aryl unit was more
readily available as the halide, which was often the case for
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Figure 11. Multiparameter optimization of potency and plasma protein binding. (top left) Fragmentation scheme used for deconstruction of known
actives. (top right) Free−Wilson model for enzyme pIC50 based on a combination of legacy (blue) and nonlegacy (red) series described in this paper.
(center right) Substructure used to filter ∼95000 potential fragment combinations down to 171. (center left) Histogram of predicted activities of these
filtered compounds, highlighting those with predicted pIC50 ≥ 10.2 in dark green. (bottom left) Scatter plot of predicted pIC50 vs predicted log K
(log(bound fraction/free fraction)). Compounds that had been synthesized prior to the analysis are shown in red, virtual compounds not synthesized
are gray, while those that were synthesized are shown in blue. The two most advanced compounds from these five are highlighted (bottom right).
a
Scheme 1
an aryl unit with the amine already in place (cf. use of 16 in
Scheme 2).
Compounds with two or three methylene units between
the aryl ring and the basic center could also be made from 13
by either route I or route II, but instead of reductive amina-
tion the base was most conveniently installed by using an
appropriate alcohol, followed by mesylation and displacement
(Scheme 5).
Synthesis of the dibasic compounds generally followed route
II, with amidation as the final step. As the binding site tolerated
only minor changes to the ortho-base with morpholine being
preferred, the second aryl ring was most often added to the core
structure with the morpholine in place. This then allowed parallel
variation of the para-base and amide (Scheme 6).
a
(a) T3P (1.57 M in THF), Et3N, 0 °C to rt, MeCN, overnight, 98%;
(b) 3-iodophenol, Cs2CO3, 60 °C, DMF, 48 h, 58%.
analogues bearing a hydroxyl on this ring (Scheme 4).
Significantly higher yields were obtained if the Suzuki reaction
was carried out using a bromoaryl aldehyde, compared to use of
H
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a
Scheme 2
a
(a) Morpholine, NaBH(OAc)3, CH2Cl2, 2 h; (b) t-BuLi, THF, −78 to 0 °C then B(Oi-Pr)3, −78 °C to rt, 1 h, then NH4Cl, H2O, 2 h, 83%
(2 steps); (c) 4 M HCl in 1,4-dioxane, CH2Cl2, 16 h, 100%; (d) 1,5-dimethyl-1H-pyrazole-3-carboxylic acid, T3P (1.57 M in THF), Et3N, MeCN,
20 h, 97%; (e) 16, SPhos, Pd(OAc)2, K2CO3, 70 °C, H2O, MeCN, 3 h, 9%.
a
Scheme 3
a
(a) 4 M HCl in 1,4-dioxane, CH2Cl2, 16 h, 100%; (b) 5-methylimidazo[1,2-a]pyridine-2-carboxylic acid, T3P (1.57 M in THF), Et3N, MeCN, 20 h,
93%; (c) 4-formylphenylboronic acid, SPhos, Pd(OAc)2, K2CO3, 80 °C, H2O, MeCN, overnight, 86%; (d) amine, NaBH(OAc)3, CH2Cl2, 1 h, 20−
78%; (e) 4-formylphenylboronic acid, SPhos, Pd(OAc)2, K2CO3, 70 °C, H2O, MeCN, 1 h, 95%; (f) cis-2,6-dimethylpiperazine, NaBH(OAc)3,
CH2Cl2, 1 h, 95%; (g) 4 M HCl in 1,4-dioxane, CH2Cl2, 16 h, 100%; (h) appropriate acid, HATU, DIPEA, MeCN, 10 min, 20−50%. Final steps
were not optimized and low yields were due to losses on HPLC purification. Compounds 10a and 10c were prepared in two steps from 17 (Scheme 2)
using route I.
Identification of Preclinical Candidates. After profiling
these ortho,para-dibasic compounds, two candidates, 12a and 12b,
emerged, showing a range of superior properties including high cell
potency, low unbound fraction in human PPB, and high in vitro
turnover in human microsomes. Both compounds also had high
solubility and could readily be crystallized to give stable crystalline
solids with one predominant polymorph. Furthermore, the crystalline
free base of 12b was investigated further and shown to have good
thermal properties (no thermal events below 80 °C) and low
hygroscopicity (<2% at 25 °C and 80% relative humidity), making it
suitable for micronization. Compounds 12a and 12b were highly
selective for PDE4 over PDE1B, PDE2A, PDE3A, PDE5, PDE6, and
PDE10A1 but showed little or no selectivity for PDE4B over PDE4D
or PDE4A, or over the high-affinity rolipram binding site (HARBS).
Both compounds were selected for in vivo profiling in rat lung
inflammation and rat and dog pharmacokinetic studies (Table 7).
Compounds 12a and 12b significantly inhibited pulmonary
inflammation with ED80 values of 0.02 and 0.06 mg/mL
nebulizer concentration, respectively (Figure 12), which
corresponded to a 0.2 and 0.6 μg/kg deposited dose in the
lung based upon measured filter sample and terminal lung levels.
In the duration experiment, both 12a and 12b inhibited LPS-
induced pulmonary neutrophilia at 0.03 and 0.1 mg/mL
nebulizer concentrations, respectively (Figure 13).
I
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a
Scheme 4
a
(a) Bis(pinacolato)diboron, (dppf)PdCl2−CH2Cl2, dppf, KOAc, 80 °C, DMSO, 20 h, 86%; (b) bromo-hydroxybenzaldehyde, either Pd(OAc)2,
SPhos, K2CO3, 80 °C, H2O, MeCN, 2 h, or Pd(PPh3)4, Na2CO3, reflux, H2O, THF, overnight, 62-73%; (c) cis-2,6-dimethylpiperazine,
NaBH(OAc)3, CH2Cl2, 2 h, 25−41%. Final step was not optimized and low yields were due to losses on HPLC purification.
a
Scheme 5
a
(a) For 21a, 20, 2-(4-bromophenyl)ethanol, for 21b−d, 17, hydroxyalkylphenylboronic acid, for 21a−d, Pd(PPh3)4, Na2CO3, reflux, H2O, THF,
overnight, 65−83%; (b) MsCl, py, CH2Cl2, overnight, 76−97%; (c) cis-2,6-dimethylpiperazine, 80 °C, microwave, MeCN, 30 min, 35−57%. Final
step was not optimized, and low yields were due to losses on HPLC purification. Compound 11e was prepared from 21b and 4-(pyrrolidin-1-
yl)piperidine using the same conditions.
a
Scheme 6
a
(a) HCHO, morpholine, 78 °C, EtOH, 11 h, 61%; (b) N-phenyltrifluoromethanesulfonimide, Et3N, 0 °C to rt, CH2Cl2, 16 h, 95%; (c)
bis(pinacolato)diboron, (dppf)PdCl2−CH2Cl2, dppf, KOAc, 80 °C, DMSO, 16 h, 97%; (d) 23, Pd(OAc)2, SPhos, K2CO3, 70 °C, H2O, MeCN, 2 h,
93%; (e) cis-2,6-dimethylpiperazine, NaBH(OAc)3, AcOH, CH2Cl2, 20 h, 96%; (f) 4 M HCl in 1,4-dioxane, CH2Cl2, 1 h, 99%; (g) appropriate acid,
EDCI, HOBt, Et3N, THF, NMP, 20 h, 43−70%.
Compounds 12a and 12b showed moderate to high clearance
in rat in vivo studies. The high terminal volumes of distribution
(Vz) for these compounds can be explained by their dibasicity
and lead to long half-lives. Compounds 12a and 12b were also
dosed via the intratracheal route, showing similar half-lives in
both plasma and lung; high concentrations were sustained in the
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Table 7. Selected Properties of 12a and 12b Compared to 8a
property
8a
12a
12b
a
human PDE4B enzyme pIC50
human PDE4D enzyme pIC50
10.8 (4)
NT
10.6 (6)
10.7 (10)
10.7 (4)
>8.0 (1)
>8.0 (1)
10.6 (6)
10.4 (12)
7.9 (10)
9.3 (2)
a
10.7 (4)
a
human PDE4A1A enzyme pIC50
NT
>8.0 (1)
a
HARBS pIC50
NT
>8.0 (1)
a
rat PDE4B enzyme pIC50
NT
10.7 (4)
a
PBMC TNF-α pIC50
10.2 (4)
NT
10.7 (2)
a
human whole blood TNF-α pIC50
9.5 (4)
a
rat whole blood TNF-α pIC50
NT
NT
a
PPB %free human/rat
5.6 (1)/10 (1)
4.5 (1)/23 (2)
>1400 (TFA salt) (1)
756/6.0/1.9 (1)
>150 (1)
10 (9)
1.3 (5)/8.3 (5)
213 (free base) (3)
765/7.1/2.8 (2)
78 (1)
a
aqueous solubility pH 7.4 (μM)
250 (free base) (1)
a
MW/clogP/log D
643/3.4/3.1 (1)
a
a
human microsomal Clint (μL/min/mg)
rat hepatocyte Clint (μL/min/106 cells)
>150 (2)
120 (2)
13 (9)
a,b
PDE selectivity
hERG binding pIC50
NT
>10000× (1)
4.7 (2)
>10000× (1)
5.2 (2)
a
4.5 (2)
a
CYP inhibition pIC50 (8 isoforms)
3A4 6.3, 2C9 6.6, 2C19 5.2, 2D6 <5 (1)
73% inhibition at 50 μM (1)
12a
all <5.0 (1)
none (1)
all <5.6 (1)
none (1)
a
time dependent CYP inhibition
8a
12b
rat
rat
dog
rat
dog
c
pharmacokinetics
Cl (mL/min/kg)
dss/Vz (L/kg)
69
44
34
50
19
V
2.6/76
13
18/27
7
26/44
15
16/30
7
13/25
15
iv t1/2 (h)
F%
NT
NT
8
16
<16
9
6
i.t. t1/2 (h)
NT
8a
12a
12b
in vivo efficacy
LPS DRC; LENC
a
0.1 mg/mL (1) 0.01 mg/mL (2)
0.01 mg/mL (2)
ED80 (nebulizer concentration/lung concentration/lung deposited dose) ND
0.02 mg/mL/23 nM/0.2 μg/kg 0.06 mg/mL/78 nM/0.6 μg/kg
a
LPS duration; LENC
0.1 mg/mL (1) 0.03 mg/mL (2)
0.1 mg/mL (2)
a
b
c
Numbers of replicates are stated in brackets. Tested against PDE1B, PDE2A, PDE3A, PDE5, PDE6, PDE10A1. n = 2−3 animals.
Figure 12. Effect of 12a and 12b on LPS-induced pulmonary neutrophilia in the rat. Both 12a (A) and 12b (B) inhibited LPS-induced neutrophilia in a
dose-dependent manner (mg/mL nebulizer concentration) when administered by aerosol nose-only inhalation of a nebulized solution 30 min before
LPS challenge. Brackets above bars signify measured terminal lung concentration (nM). n = 8 in all groups except n = 4 for saline group. Data
representative from two separate experiments. *, **, *** = p < 0.05, 0.01, and 0.001 respectively.
lung while plasma concentrations were significantly lower,
thereby offsetting the potential for any adverse side effects
(Figure S4, Supporting Information). These compounds also
showed low oral exposure and predictable t1/2 in dog, consistent
with the low fraction absorbed, high clearance, and Vz observed
in the rat. Interestingly, there were no emetic episodes noted
during either the iv or po PK studies in dog with 12b. The dose
equated to an iv Cmax(free) of 8 nM (iv dose 0.1 mg/kg), equating
to a Cmax(free)/PBMC potency ratio of 280, which gave confidence
that emesis should not be an issue in the clinic for this compound.
A simple, pragmatic approach to predict human dose was to
use the dose required to give 80% inhibition efficacy in the rat
acute LPS challenge model and adjust for human−rat differences
in the PDE4 binding potency. As the estimated lung delivered
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Figure 13. Effect of 12a and 12b when dosed 12 h before LPS exposure in the rat. Both 12a (A) and 12b (B) inhibited LPS-induced neutrophilia when
administered by aerosol nose-only inhalation of a nebulized solution 12 h before LPS challenge (mg/mL nebulizer concentration). Brackets above bars
signify measured terminal lung concentration (nM). n = 8 in all groups except n = 4 for saline group. Data representative from two separate experiments.
*, **, *** = p < 0.05, 0.01, and 0.001, respectively.
doses for 12b and 12a were 0.6 and 0.2 μg/kg, respectively, and
PDE4 potencies in rat and human were equivalent, the expected
human doses for compounds 12b and 12a were predicted to be
0.6 and 0.2 μg/kg, respectively (Table 8). A complementary yet
(Figures S6 and S7, Supporting Information).20 Finally the
simulated human terminal lung half-lives were consistent with
these compounds being suitable for once daily dosing.
Experiments to investigate the emetic profile of 12b were
undertaken in ferrets.31 The compound was administered by iv
infusion (0.3, 1, and 3 mg/kg), then the animals were observed
for incidence of emetic episodes, salivation, and behavioral
changes. 12b shows a 625× free plasma/PBMC ratio at the non-
nauseous dose of 0.3 mg/kg. At doses which produced retching
in 50% of the animals, the ratio of free plasma/PBMC potency
was 243× higher for 12b compared to 2, demonstrating
that 12b has a much lower emetic potential. Given that the
predicted efficacious human inhaled dose for 12b is so low, it is
highly unlikely that this compound would cause emesis in the
clinic.
Table 8. Predicted Human Pharmacokinetics for 12a and 12b
parameter
12a 12b
human total clearance (mL/min/kg)
9
3
human t1/2 (h)
14
10
lung delivered dose predicted from the rat LPS model after
adjusting for species differences in potency (μg/kg)
0.2 0.6
predicted lung delivered dose using PK modeling method (μg/kg)
1
1
more robust approach was to predict the human dose from a
multicompartment PK model (using a nonlinear regression
model in WinNonlin) as described previously.30 This model
represents blood, tissues, and a “deep compartment”, hypothe-
sized to consist of specific tissues having higher affinity for dibasic
compounds, perhaps in organelles such as lysosomes. It was
speculated that the terminal half-lives were controlled largely by
the rate of diffusion of compounds from this deep compartment
back into blood. A range of reference compounds were dosed to
rat via both iv and i.t. routes, and iv route in dog, and both plasma
and lung measurements were made over time. Furthermore, both
plasma and lung measurements were determined after inhalation
dosing in rat. The lung and plasma concentration−time PK data
for 12a and 12b were fitted to this five-compartmental model
which described the profiles well. The therapeutic human dose
was estimated from the predicted lung concentration at the end
of the dosing interval. This was possible because it was known
what lung concentration was required for 80% efficacy in the
rat acute LPS model. The ED80s were calculated at 0.02 and
0.06 mg/mL nebulizer concentrations respectively for 12a and
12b, which gave total lung concentrations of 23 and 78 nM, and it
can be assumed that the same lung concentration in man will
give equivalent efficacy. Clearance was estimated to be 9 and
3 mL/min/kg (Figure S5, Supporting Information), plasma
fraction unbound was measured as 0.045 and 0.013, and
physiological lung volume was taken to be 8 mL/kg. This
simulation estimated an efficacious human lung deposited dose
of 1 μg/kg for both compounds, which was comparable to the
lung dose required in the rat LPS model. The total Cmax,ss were
also comparable to the values estimated from the correlation
between dose and Cmax with other marketed inhaled therapies
CONCLUSIONS
■
We have reported a lead optimization campaign that has led to
the discovery of novel and highly potent inhaled PDE4
inhibitors. Combining both learnings and shortcomings from
the medicinal chemistry properties associated with 4 and our
own historical series, a general method to confidently predict
whether a compound would show efficacy and duration in our in-
house efficacy experiment was developed using nominal logistic
regression. Toward the end of the lead optimization process, a
Free−Wilson model was built for PDE4 potency and a large
virtual library of compounds were proposed. Compounds were
prioritized using this model and human protein binding
predictions and a focused synthesis campaign led to compounds
12a and 12b. These both showed striking anti-inflammatory
efficacy when administered by inhalation to rats as aqueous
solutions either 30 min or up to 12 h before LPS challenge. They
showed sustained duration of action in these in-house models
which was superior to that of 4 and 5. Compound 12b had a very
low predicted efficacious human inhaled dose of 1 μg/kg and
would be highly unlikely to cause emesis in the clinic due to its
very high ratio of free plasma/PBMC potency compared to
cilomilast in ferret emesis experiments. Compounds 12a and 12b
thus serve as interesting preclinical compounds for the treatment
of COPD.
EXPERIMENTAL SECTION
Reagents were obtained from commercial suppliers and used without
purification. Unless otherwise stated, reactions were carried out at
■
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ambient temperature (18−25 °C) and under positive nitrogen pressure
with magnetic stirring. Flash chromatography was performed on
E. Merck 230−400 mesh silica gel 60 or by using Biotage KP-Sil SNAP
cartridges. Solvent mixtures used as eluents are volume/volume ratios.
Preparative HPLC purifications were performed on Waters Symmetry,
Novapak, or XTerra columns eluting with a gradient of acetonitrile or
methanol in aqueous ammonium acetate, ammonia, or trifluoroacetic
N-((1s,4s)-4-Aminocyclohexyl)-5-fluoro-2-(3-iodophenoxy)-
nicotinamide 26 [General Method A]. To a solution of 13 (3.10 g,
1 equiv) in CH2Cl2 (30 mL) was added 4 M HCl in 1,4-dioxane (20 mL,
14 equiv). The mixture was stirred at rt overnight then concentrated to
1
dryness to give 26 hydrochloride as a white solid (2.74 g, 100%). H
NMR (400 MHz, CD3OD, δ): 8.10 (d, J = 3.1 Hz, 1H), 8.02 (dd, J = 7.9,
3.1 Hz, 1H), 7.61−7.57 (m, 2H), 7.19−7.17 (m, 2H), 4.13−4.08 (m,
1H), 3.27−3.22 (m, 1H), 1.97−1.65 (m, 8H). This compound could
also be used as the free base after neutralization and extraction into
CH2Cl2.
1
acid solution. Routine H NMR spectra were recorded on a Varian
UnityInova spectrometer at a proton frequency of 300 or 400 MHz or a
Bruker Avance III spectrometer at a proton frequency of 500 MHz.
Chemical shifts are expressed in ppm. Mass spectra were measured on an
Agilent 1100 MSD G1946D spectrometer using electrospray ionization,
atmospheric pressure chemical ionization, or Agilent Multimode
ionization; only ions that indicate the parent mass are reported. The
purity of all test compounds was determined by RP HPLC conducted on
an Agilent 1100 LC/MS system, using UV detection and a gradient of
5−95% acetonitrile in either aqueous ammonium acetate (0.1% w/v) or
trifluoroacetic acid (0.1% v/v) over 2.5 min on 2.1 mm × 5 mm columns
packed with Waters Symmetry C8 or Waters Symmetry C18, or
2.0 mm × 50 mm columns packed with Phenomenex Max-RP. All test
compounds were ≥95% purity.
tert-Butyl (1s,4s)-4-(2-Chloro-5-fluoronicotinamido)-
cyclohexylcarbamate 25. 2-Chloro-5-fluoronicotinic acid 14 (16.5 g,
1 equiv) and tert-butyl (1s,4s)-4-aminocyclohexylcarbamate 15 (25.2 g,
1.25 equiv) were suspended in MeCN (550 mL), then Et3N (78 mL,
6 equiv) was added. The stirred mixture was cooled in ice while T3P
(1.57 M in THF, 78 mL, 1.3 equiv) was slowly added. The reaction became
homogeneous and was allowed to slowly warm to rt overnight. All volatiles
were evaporated, and the residue was partitioned between aqueous 2 M
HCl and EtOAc. The aqueous phase was separated and extracted into
further EtOAc (×2). The combined organic solutions were washed with
water, saturated aqueous NaHCO3, and brine. The organic layer was dried
(MgSO4) and concentrated to give 25 as a cream-colored foam (34.2 g,
98%). 1H NMR (400 MHz, CDCl3, δ): 8.34 (d, J = 2.8 Hz, 1H), 7.92 (dd,
J = 7.9, 3.1 Hz, 1H), 6.70(d, J = 6.7Hz, 1H), 4.56 (d, J = 6.4 Hz, 1H), 4.21−
4.12 (m, 1H), 3.71−3.57 (m, 1H), 1.92−1.80 (m, 4H), 1.79−1.70 (m, 2H),
1.64−1.50 (m, 2H), 1.45 (s, 9H).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(3-iodophenoxy)nicotinamide 17 [Gen-
eral Method B]. T3P (1.57 M in THF, 5.2 mL, 1.1 equiv) was added to
a stirred solution of 26 (3.50 g, 1 equiv), 1,5-dimethyl-1H-pyrazole-3-
carboxylic acid (1.10 g, 1.1 equiv), and Et3N (9.9 mL, 10 equiv) in
MeCN (20 mL) and stirred at rt for 20 h. The mixture was concentrated,
dissolved in EtOAc, washed with saturated aqueous NaHCO3, water,
and brine, dried (MgSO4), and concentrated to give 17 as a white solid
(4.00 g, 97%). 1H NMR (400 MHz, CDCl3, δ): 8.36 (dd, J = 8.2, 3.1 Hz,
1H), 8.07 (d, J = 3.1 Hz, 1H), 7.87 (d, J = 6.7 Hz, 1H), 7.64 (dt, J =
7.5, 1.5 Hz, 1H), 7.55 (t, J = 1.8 Hz, 1H), 7.22−7.14 (m, 2H), 6.69 (d, J =
7.7 Hz, 1H), 6.53 (s, 1H), 4.26−4.17 (m, 1H), 4.12−4.03 (m, 1H),
3.78 (s, 3H), 2.28 (s, 3H), 1.95−1.73 (m, 6H), 1.67−1.58 (m, 2H).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(4′-hydroxy-2′-(morpholinomethyl)-
biphenyl-3-yloxy)nicotinamide 8a. To a solution of SPhos (28 mg,
0.2 equiv) in MeCN (15 mL) was added Pd(OAc)2 (8 mg, 0.1 equiv).
The mixture was stirred for 10 min at rt before a solution of K2CO3
(0.144 g, 3 equiv) in water (5 mL) was added, followed by 17 (0.200 g,
1 equiv) and 16 (82 mg, 1 equiv). The mixture was heated at 70 °C for
3 h then poured into water and extracted into EtOAc, and the crude
product was purified by preparative HPLC to give 8a as a solid (21 mg,
9%). 1H NMR (400 MHz, CDCl3, δ): 8.33 (dd, J = 7.9, 3.1 Hz, 1H), 8.05
(d, J = 3.1 Hz, 1H), 8.02 (d, J = 7.2 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.31
(s, 1H), 7.23−7.18 (m, 3H), 7.14 (d, J = 7.7 Hz, 1H), 7.10 (t, J = 1.8 Hz,
1H), 6.90 (dd, J = 8.5, 2.1 Hz, 1H), 6.51 (s, 1H), 4.30 (s, 2H), 4.24−4.19
(m, 1H), 4.06−4.00 (m, 1H), 3.83−3.77 (m, 4H), 3.73 (s, 3H), 3.22−
2.72 (m, 4H), 2.29 (s, 3H), 1.94−1.79 (m, 6H), 1.71−1.62 (m, 2H).
LRMS: m/z 643 [M + H]+.
tert-Butyl (1s,4s)-4-(5-Fluoro-2-(3-iodophenoxy)-
nicotinamido)cyclohexylcarbamate 13. A mixture of 25 (11.31 g,
1 equiv), 3-iodophenol (6.69 g, 1 equiv), and Cs2CO3 (19.82 g, 2 equiv)
was heated at 60 °C in DMF (150 mL) for 48 h then poured onto water
(300 mL). The resulting solid was removed by filtration and dissolved in
EtOAc, which was washed with aqueous 2 M HCl, water, and brine. The
organic layer was dried (Na2SO4) and concentrated to give crude product,
which was purified by flash silica chromatography, elution gradient 30−
40% EtOAc in isohexane, to give 13 as a white foam (9.80 g, 58%). 1H
NMR (300 MHz, CDCl3, δ): 8.35 (dd, J = 8.2, 3.2 Hz, 1H), 8.06 (d, J = 3.3
Hz, 1H), 7.84 (d, J = 7.1 Hz, 1H), 7.65 (dt, J = 7.6, 1.4 Hz, 1H), 7.53 (t, J =
1.7 Hz, 1H), 7.23−7.11 (m, 2H), 4.41 (s, 1H), 4.21−4.10 (m, 1H), 3.59−
3.67 (m, 1H), 1.87−1.75 (m, 6H), 1.75−1.62 (m, 2H), 1.44 (s, 9H).
4-Hydroxy-2-(morpholinomethyl)phenylboronic Acid 16.
Morpholine (1.19 mL, 1.1 equiv) was added to a solution of
2-bromo-5-hydroxybenzaldehyde (2.50 g, 1 equiv) in CH2Cl2 (20 mL)
and stirred for 20 min. Sodium triacetoxyborohydride (2.90 g, 1.1 equiv)
was added and stirred for 2 h. The reaction was quenched with MeOH
and stirred for 1 h. The solution was concentrated, dissolved in MeOH,
and loaded onto a SCX (50 g) column, flushing with MeOH. Eluting
with methanolic ammonia and concentration of the eluent gave
4-bromo-3-(morpholinomethyl)phenol (3.0 g, 89%), which was
dissolved in THF (60 mL) and cooled to −78 °C, then 1.7 M
t-butyllithium (19.45 mL, 3 equiv) was added dropwise. The reaction
mixture was stirred for 10 min then warmed to 0 °C for 15 min. The
reaction was then cooled to −78 °C and triisopropyl borate (7.6 mL,
3 equiv) added. The reaction was warmed to rt and stirred for 1 h.
Saturated aqueous NH4Cl was added and the reaction stirred at rt for
2 h. The reaction mixture was diluted with EtOAc (200 mL), the phases
were separated, and the aqueous was further extracted with EtOAc. The
combined organic extracts were washed with saturated brine, dried
(MgSO4), and concentrated to give 16 (2.45 g, 83% over 2 steps).
LRMS: m/z 238 [M + H]+.
tert-Butyl (1s,4s)-4-(5-Fluoro-2-(4′-formylbiphenyl-3-yloxy)-
nicotinamido)cyclohexylcarbamate 27 [General Method C]. A
solution of K2CO3 (1.64 g, 3 equiv) in water (15 mL), 13 (2.20 g,
3 equiv), and 4-formylphenylboronic acid (0.713 g, 1.2 equiv) were
added sequentially to a stirred solution of Pd(OAc)2 (89 mg, 0.1 equiv)
and SPhos (0.319 g, 0.2 equiv) in MeCN (20 mL) then heated at 70 °C
for 1 h. The mixture was cooled to rt, extracted with EtOAc, washed with
water and brine, dried (MgSO4), and concentrated. The residue was
purified by flash silica chromatography, eluting with 40% EtOAc in
1
isohexane to give 27 (2.00 g, 95%). H NMR (300 MHz, CDCl3, δ):
10.01 (s, 1H), 8.33 (dd, J = 7.9, 3.3 Hz, 1H), 8.03 (d, J = 3.3 Hz, 1H),
7.92 (d, J = 7.9 Hz, 2H), 7.73 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 5.3 Hz,
2H), 7.41 (s, 1H), 7.18 (m, 1H), 4.48 (m, 1H), 4.16 (m, 1H), 3.58 (m,
1H), 1.75 (m, 6H), 1.50 (m, 2H), 1.36 (s, 9H).
tert-Butyl (1s,4s)-4-(2-(4′-(((3S,5R)-3,5-Dimethylpiperazin-1-
yl)methyl)biphenyl-3-yloxy)-5-fluoronicotinamido)-
cyclohexylcarbamate 28 [General Method D]. To a solution of
27 (0.780 g, 1 equiv) in CH2Cl2 (5 mL) was added cis-2,6-dimethyl-
piperazine (0.250 g, 1.5 equiv). The mixture was allowed to stir at rt for
2 h, then sodium triacetoxyborohydride (0.465 g, 1.5 equiv) was added.
After stirring for 1 h, the mixture was diluted with CH2Cl2 and washed
with saturated aqueous NaHCO3, dried (MgSO4), and concentrated
to give 28 as a foam (0.880 g, 95%). 1H NMR (300 MHz, CDCl3, δ):
8.37 (dd, J = 7.8, 3.1 Hz, 1H), 8.08−8.03 (m, 2H), 7.56−7.53 (m, 4H),
7.42−7.35 (m, 3H), 7.15−7.10 (m, 1H), 4.38−4.31 (m, 1H),
4.22−4.14 (m, 1H), 3.65−3.57 (m, 1H), 3.54 (s, 2H), 3.00−2.93
(m, 2H), 2.82−2.75 (m, 2H), 1.85−1.61 (m, 10H), 1.41 (s, 9H), 1.04
(d, J = 6.4 Hz, 6H).
tert-Butyl (1s,4s)-4-(5-Fluoro-2-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenoxy)nicotinamido)cyclohexylcarbamate
29 [General Method E]. PdCl2(dppf)CH2Cl2 (0.129 g, 0.05 equiv)
and dppf (88 mg, 0.05 equiv) were stirred together in dry DMSO
M
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(4 mL) for 10 min, then KOAc (0.927 g, 3 equiv), a solution of 13
(1.748 g, 1 equiv) in DMSO (8 mL), and bis(pinacolato)diboron (1.063 g,
1.3 equiv) were added and the reaction mixture heated at 80 °C for 16 h.
After cooling to rt, water (10 mL) was added and the mixture stirred for 3 h.
The solid was removed by filtration and washed with water (2 × 10 mL)
then purified by flash silica chromatography, elution gradient 60−100%
EtOAc in isohexane to give 29 as a pale-brown oil (1.704 g, 97%). 1H NMR
(400 MHz, CDCl3, δ): 8.35 (dd, J = 8.2, 3.1 Hz, 1H), 8.06−8.03 (m, 2H),
7.76 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 2.6 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H),
7.28−7.25 (m, 1H), 4.44−4.38 (m, 1H), 4.19−4.14 (m, 1H), 3.65−3.59
(m, 1H), 1.87−1.36 (m, 8H), 1.42 (s, 9H), 1.35 (s, 12H).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenoxy)nicotinamide 20. Prepared from 17 using general
method E, in 86% yield. 1H NMR (400 MHz, CDCl3, δ): 8.36 (dd, J =
8.3, 3.2 Hz, 1H), 8.10−8.04 (m, 2H), 7.74 (dd, J = 6.4, 1.0 Hz, 1H), 7.56
(d, J = 2.3 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.29 (ddd, J = 8.1, 2.5, 1.1
Hz, 1H), 6.65 (d, J = 7.4 Hz, 1H), 6.51 (s, 1H), 4.26−4.17 (m, 1H),
4.11−4.03 (m, 1H), 3.75 (s, 3H), 2.27 (s, 3H), 1.94−1.73 (m, 6H),
1.67−1.56 (m, 2H), 1.32 (s, 12H).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(4′-(3-hydroxypropyl)biphenyl-3-
yloxy)nicotinamide 21a [General Method F]. Pd(PPh3)4 (24 mg,
0.02 equiv) was added to a mixture of 17 (0.600 g, 1 equiv), 4-(3-
hydroxypropyl)phenylboronic acid (0.224 g, 1.2 equiv), and Na2CO3
(0.330 g, 3 equiv) in water (2.5 mL) and THF (5 mL). The mixture was
heated at 70 °C for 18 h, then allowed to cool, diluted with EtOAc, and
washed with water and brine. The organic layer was dried (Na2SO4) and
concentrated to give crude product which was purified by flash silica
chromatography, eluting with 0−5% MeOH in EtOAc to give 21a as a
white foam (0.480 g, 79%). 1H NMR (400 MHz, CD3OD, δ): 8.37 (dd,
J = 8.2, 3.1 Hz, 1H), 8.11−8.05 (m, 2H), 7.55−7.47 (m, 4H), 7.37−7.35
(m, 1H), 7.25−7.22 (m, 2H), 7.16−7.12 (m, 1H), 6.67 (d, J = 7.7 Hz,
1H), 6.50 (s, 1H), 4.27−4.19 (m, 1H), 4.12−4.01 (m, 1H), 3.70 (t, J =
6.3 Hz, 2H), 3.66 (s, 3H), 2.74 (t, J = 7.8 Hz, 2H), 2.25 (s, 3H) 1.95−
1.75 (m, 6H), 1.67−1.54 (m, 4H).
using general method G, as the trifluoroacetic acid salt in 30% yield over
2 steps. 1H NMR (400 MHz, DMSO-d6, 90 °C, δ): 8.21 (d, J = 3.1 Hz,
1H), 8.12−8.07 (m, 1H), 8.03−7.95 (m, 1H), 7.63−7.55 (m, 2H),
7.51−7.44 (m, 2H), 7.44−7.40 (m, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.22−
7.14 (m, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.38−6.33 (m, 1H), 4.03−3.95
(m, 1H), 3.88−3.77 (m, 1H), 3.70 (s, 3H), 3.58−3.08 (m, 11H), 3.02−
2.91 (m, 2H), 2.91−2.70 (m, 2H), 2.24 (s, 3H), 2.02−1.61 (m, 14H).
LRMS: m/z 708 [M + H]+.
4-Hydroxy-3-(morpholinomethyl)benzaldehyde 22. 4-
Hydroxybenzaldehyde (10.0 g, 1 equiv) was dissolved in ethanol
(100 mL) and formaldehyde (37% in water, 6.1 mL, 1 equiv) added,
followed by morpholine (7.1 mL, 1 equiv). The reaction mixture was
heated at 78 °C for 6 h, left at rt overnight, then further additions of 37%
aqueous formaldehyde and morpholine (0.5 eq of each) were made
and the mixture heated at 78 °C for 5 h. The mixture was concen-
trated and the residue purified by flash silica chromatography, elution
gradient 20−50% EtOAc (containing 2% Et3N and 2% MeOH)
1
in isohexane to give 22 as a colorless oil (11.00 g, 61%). H NMR
(500 MHz, CDCl3, δ): 9.82 (s, 1H), 7.72 (dd, J = 8.3, 1.9 Hz, 1H), 7.58
(s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.85−3.70 (m, 4H), 3.80 (s, 2H),
2.70−2.50 (m, 4H).
4-Formyl-2-(morpholinomethyl)phenyl Trifluoromethane-
sulfonate 23. N-Phenyltrifluoromethanesulfonimide (5.09 g, 1.5 equiv)
was added to an ice-cooled solution of 22 (2.10 g, 1 equiv) and Et3N
(4 mL, 3 equiv) in CH2Cl2 (20 mL). The mixture was allowed to warm
to rt and stirred for a further 16 h. The solution was washed with water
and brine, dried (Na2SO4), and concentrated. The residue was purified
by flash silica chromatography eluting with 30% EtOAc in isohexane to
give 23 as a colorless oil (3.17 g, 95%). 1H NMR (400 MHz, CDCl3, δ):
10.05 (s, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.90 (dd, J = 8.3, 2.2 Hz, 1H),
7.44 (d, J = 8.4 Hz, 1H), 3.74−3.68 (m, 4H), 3.62 (s, 2H), 2.51−
2.42 (m, 4H).
tert-Butyl (1s,4s)-4-(5-Fluoro-2-(4′-formyl-2′-(morpholinomethyl)-
biphenyl-3-yloxy)nicotinamido)cyclohexylcarbamate 24. A solution
of K2CO3 (0.396 g, 3 equiv) in water (3 mL), 23 (0.405 g, 1.2 equiv) and
29 (0.530 g, 1 equiv) were added sequentially to a stirred solution
of Pd(OAc)2 (21 mg, 0.1 equiv) and SPhos (78 mg, 0.2 equiv) in MeCN
(5 mL). The mixture was heated at 70 °C for 2 h then cooled to rt,
extracted with EtOAc, washed with water and brine, dried (MgSO4), and
concentrated. The residue was purified by flash silica chromatography
eluting with 30% EtOAc in isohexane to give 24 as a brown foam
(0.560 g, 93%). 1H NMR (400 MHz, CDCl3, δ): 10.07 (s, 1H), 8.37
(dd, J = 8.2, 3.1 Hz, 1H), 8.06 (d, J = 3.1 Hz, 1H), 8.01 (d, J = 1.5 Hz,
1H), 7.96 (d, J = 7.3 Hz, 1H), 7.85 (dd, J = 7.8, 1.7 Hz, 1H), 7.59−
7.46 (m, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.32−7.29 (m, 1H), 7.23−7.17
(m, 1H), 4.56−4.41 (m, 1H), 4.22−4.13 (m, 1H), 3.67−3.60 (m, 1H),
3.60−3.55 (m, 4H), 3.49 (s, 2H), 2.41−2.30 (m, 4H), 1.89−1.47
(m, 8H), 1.43 (s, 9H).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-2-(4′-(3-((3S,5R)-3,5-dimethylpiperazin-1-yl)-
propyl)biphenyl-3-yloxy)-5-fluoronicotinamide 11a [General
Method G]. To a solution of 21a (0.610 g, 1 equiv) and pyridine
(0.30 mL, 3.5 equiv) in CH2Cl2 (7 mL) was added mesyl chloride
(0.28 mL, 3.5 equiv) and the reaction stirred at rt overnight. The
reaction mixture was concentrated to give a residue which was
partitioned between EtOAc and aqueous 2 M HCl. The organic layer
was washed with further 2 M HCl, saturated aqueous NaHCO3, and
brine, dried (MgSO4), and concentrated to give a yellow oil. This
was triturated with ether to give the methanesulfonate as a white solid
(0.665 g, 96%). The methanesulfonate (0.145 g, 1 equiv) was added to a
microwave tube with cis-2,6-dimethylpiperazine (75 mg, 3 equiv) and
MeCN (1 mL). The reaction was microwaved at 80 °C for 30 min.
The crude product was purified by preparative HPLC to give
tert-Butyl (1s,4s)-4-(2-(4′-(((3S,5R)-3,5-Dimethylpiperazin-1-
yl)methyl)-2′-(morpholinomethyl)biphenyl-3-yloxy)-5-
fluoronicotinamido)cyclohexylcarbamate 30. 24 (0.280 g, 1
equiv) and cis-2,6-dimethylpiperazine (51 mg, 1 equiv) were stirred in
CH2Cl2 (10 mL) for 15 min. AcOH (51 μL, 2 equiv) was added,
followed by sodium triacetoxyborohydride (0.188 g, 2 equiv), and the
reaction stirred for a further 20 h. The reaction was quenched with
aqueous 2 M HCl, extracted with EtOAc, washed with water and brine,
dried (Na2SO4), and concentrated to give 30 as a light-brown foam
1
11a trifluoroacetic acid salt as a white solid (86 mg, 43%). H NMR
(400 MHz, CD3OD, δ): 8.13−8.07 (m, 1H), 8.04 (d, J = 7.9 Hz, 1H),
7.54−7.36 (m, 5H), 7.24 (d, J = 7.2 Hz, 2H), 7.18−7.09 (m, 1H), 6.43
(s, 1H), 4.13−4.05 (m, 1H), 3.97−3.87 (m, 1H), 3.75−3.55 (m, 7H),
3.11−2.98 (m, 2H), 2.90−2.76 (m, 2H), 2.77−2.65 (m, 2H), 2.24
(s, 3H), 2.10−1.97 (m, 2H), 1.91−1.59 (m, 8H), 1.35 (d, J = 6.0 Hz,
6H). LRMS: m/z 682 [M + H]+.
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(4′-(2-hydroxyethyl)biphenyl-3-yloxy)-
nicotinamide 21b. Prepared from 20 and 2-(4-bromophenyl)ethanol
using general method F, in 70% yield. 1H NMR (400 MHz, CDCl3, δ):
8.38 (dd, J = 8.2, 3.1 Hz, 1H), 8.08 (d, J = 3.1 Hz, 1H), 8.06 (s, 1H),
7.56−7.48 (m, 4H), 7.38−7.34 (m, 1H), 7.29−7.26 (m, 2H), 7.17−7.13
(m, 1H), 6.67 (d, J = 7.5 Hz, 1H), 6.50 (s, 1H), 4.27−4.19 (m, 1H),
4.12−4.01 (m, 1H), 3.94−3.86 (m, 2H), 3.67 (s, 3H), 2.90 (t, J = 6.5 Hz,
2H), 2.25 (s, 3H), 1.96−1.75 (m, 6H), 1.68−1.57 (m, 2H).
1
(0.310 g, 96%). H NMR (300 MHz, CDCl3, δ): 8.36 (dd, J = 8.2,
3.1 Hz, 1H), 8.06 (d, J = 3.2 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.50 (t,
J = 7.9 Hz, 1H), 7.40−7.27 (m, 5H), 7.19−7.11 (m, 1H), 4.57−4.41
(m, 1H), 4.23−4.10 (m, 1H), 3.68−3.59 (m, 1H), 3.59−3.50 (m, 6H),
3.42 (s, 2H), 3.09−2.95 (m, 2H), 2.74−2.85 (m, 2H), 2.40−2.27
(m, 4H), 1.90−1.59 (m, 8H), 1.58−1.46 (m, 2H), 1.42 (s, 9H), 1.10
(d, J = 6.3 Hz, 6H).
N-((1s,4s)-4-Aminocyclohexyl)-2-(4′-(((3S,5R)-3,5-dimethyl-
piperazin-1-yl)methyl)-2′-(morpholinomethyl)biphenyl-3-
yloxy)-5-fluoronicotinamide 31. To a stirred solution of 30 (0.300 g,
1 equiv) in CH2Cl2 (1 mL) was added 4 M HCl in 1,4-dioxane (1 mL,
10 equiv). The reaction mixture was stirred at rt for 1 h then
concentrated to give 31 as the trihydrochloride salt (0.300 g, 99%).
N-((1s,4s)-4-(1,5-Dimethyl-1H-pyrazole-3-carboxamido)-
cyclohexyl)-5-fluoro-2-(4′-(2-(4-(pyrrolidin-1-yl)piperidin-1-yl)-
ethyl)biphenyl-3-yloxy)nicotinamide 11e. Prepared from 21b and
4-(pyrrolidin-1-yl)piperidine via the intermediate methanesulfonate
N
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1H NMR (400 MHz, DMSO-d6, δ): 8.28 (d, J = 7.5 Hz, 1H), 8.24 (d,
J = 3.1 Hz, 1H), 8.02 (dd, J = 8.1, 3.1 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H),
7.39 (s, 1H), 7.35−7.38 (m, 1H), 7.22−7.28 (m, 3H), 7.19 (ddd, J =
8.2, 2.4, 0.9 Hz, 1H), 3.94−3.86 (m, 1H), 3.47−3.42 (m, 6H), 3.35
(s, 2H), 2.80−2.68 (m, 3H), 2.68−2.61 (m, 2H), 2.29−2.21 (m, 4H),
1.78−1.64 (m, 2H), 1.62−1.44 (m, 6H), 1.43−1.31 (m, 2H), 0.88 (d,
J = 6.3 Hz, 6H).
ACKNOWLEDGMENTS
■
We acknowledge Pat Barton, Fiona Bell, Amanda Benjamin,
Roger Bonnert, Juan Carrillo, John Collington, Rebecca Denton,
Theresa Humphries, Fraser Hunt, Ray Hutchinson, Tony Ingall,
Robert Jewell, Andy Kirk, Sarah Lever, Dermot McGinnity,
Premji Meghani, Sandy Nicol, Victor Oreffo, Austen Pimm,
Aaron Rigby, Andrew Robbins, Joanne Ross, Hitesh Sanganee,
and Jerzy Schmidt.
2-(4′-(((3S,5R)-3,5-Dimethylpiperazin-1-yl)methyl)-2′-
(morpholinomethyl)biphenyl-3-yloxy)-5-fluoro-N-((1s,4s)-4-(2-
hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide 12b
[General Method H]. HOBt (0.473 g, 2 equiv), EDCI (0.592 g,
2 equiv), and Et3N (0.215 mL, 1 equiv) were added to a solution of
2-hydroxy-5-methylbenzoic acid (0.470 g, 2 equiv) in THF (5 mL). The
mixture was stirred for 10 min at rt then added to a solution of 31
hydrochloride (1.200 g, 1 equiv) and Et3N (0.861 mL, 4 equiv) in THF
(5 mL) and NMP (5 mL). The reaction mixture was stirred at rt
overnight then evaporated to dryness and the residue dissolved in
CH2Cl2 (150 mL) and washed with saturated aqueous NaHCO3 and
water, dried (MgSO4), and concentrated. The crude product was
purified by preparative HPLC and then by flash silica chromatography
eluting with 4% 7 M NH3 in MeOH in CH2Cl2 to give 12b as a white
ABBREVIATIONS USED
■
dppf, 1,1′-bis(diphenylphosphino)ferrocene; BAL, bronchoal-
veolar lavage; SPhos, 2-dicyclohexylphosphino-2′,6′-dimethoxy-
biphenyl; DIPEA, N,N-diisopropylethylamine; EDCI, N-(3-
(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride;
HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-
1-ylmethylene]-N-methylmethanaminium hexafluorophosphate
N-oxide; DRC, dose−response curve; ED80, effective dose giving
80% inhibition; HOBt, 1-hydroxybenzotriazole hydrate; i.t.,
intratracheal instillation; LENC, lowest effective nebulizer
concentration; ND, not determined; NT, not tested; PBMC,
peripheral blood mononuclear cell; PDE4, phosphodiesterase 4;
T3P, propylphosphonic anhydride; RP HPLC, reverse phase
HPLC; RMSE, root-mean-square error; SCX, strong cation
exchange chromatography
1
solid (0.505 g, 43%). H NMR (400 MHz, CD3OD, δ): 8.10 (d, J =
3.1 Hz, 1H), 8.05 (dd, J = 8.0, 3.1 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.43
(t, J = 7.9, 7.9 Hz, 1H), 7.38 (s, 1H), 7.28−7.24 (m, 1H), 7.23−7.13 (m,
4H), 7.10 (dd, J = 8.4, 2.2 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 4.14−4.06
(m, 1H), 4.04−3.94 (m, 1H), 3.52 (s, 2H), 3.50−3.44 (m, 4H), 3.38
(s, 2H), 2.98−2.85 (m, 2H), 2.84−2.73 (m, 2H), 2.31−2.15 (m, 7H),
1.90−1.62 (m, 10H), 1.03 (d, J = 6.4 Hz, 6H). LRMS: m/z 765 [M + H]+
N-((1s,4s)-4-(2-(4′-(((3S,5R)-3,5-Dimethylpiperazin-1-yl)-
methyl)-2′-(morpholinomethyl)biphenyl-3-yloxy)-5-
fluoronicotinamido)cyclohexyl)-4-methylthiazole-2-carboxa-
mide 12a. Prepared from 4-methylthiazole-2-carboxylic acid and 31
hydrochloride using general method H, as the trifluoroacetic acid salt in
70% yield. 1H NMR (400 MHz, CD3OD, δ): 8.14 (d, J = 3.1 Hz, 1H),
8.04 (dd, J = 7.9, 3.1 Hz, 1H), 7.74 (d, J = 1.4 Hz, 1H), 7.58 (t, J = 7.9 Hz,
1H), 7.51 (dd, J = 7.9, 1.5 Hz, 1H), 7.41−7.36 (m, 2H), 7.32 (dd, J = 8.0,
2.1 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.18−7.15 (m, 1H), 4.43 (s, 2H),
4.15−4.08 (m, 1H), 4.02−3.93 (m, 1H), 3.87−3.64 (m, 6H), 3.49−3.38
(m, 2H), 3.26−2.72 (m, 6H), 2.44 (s, 3H), 2.32−2.21 (m, 2H), 1.94−
1.73 (m, 8H), 1.29 (d, J = 6.6 Hz, 6H). LRMS: m/z 756 [M + H]+.
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S
* Supporting Information
Further synthetic details and full descriptions of biological
methods used. This material is available free of charge via the
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AUTHOR INFORMATION
■
Corresponding Authors
*For C.D.S.: phone +1 781-228-0743; e-mail, chris.desavi2@
astrazeneca.com.
*For R.J.C.: phone +46 31 77 61794; e-mail, rhona.cox@
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Author Contributions
C.D.S., R.J.C., A.R.C., M.R.D., A.M., L.C.M., and B.P. performed
medicinal and synthetic chemistry. D.J.W. carried out modeling
and property predictions. G.A., S.S.Y., P.S.G., and M.S.D. carried
out biological profiling. R.R. carried out pharmacokinetic
profiling. All authors contributed to the writing of this
manuscript and have given approval to the final version.
Notes
All animal studies were carried out under licenses issued under
the Animals (Scientific Procedures) Act 1986 following local
ethical committee review.
The authors declare no competing financial interest.
O
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Journal of Medicinal Chemistry
Article
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P. T.; Facchinetti, F.; Carnini, C.; Moretto, N.; Patacchini, R.; Bassani,
F.; Cenacchi, V.; Volta, R.; Amadei, F.; Capacchi, S.; Delcanale, M.;
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