Design of Phthalazinone Amide Histamine H1 Receptor Antagonists for Use in Rhinitis

Article abstract of DOI:10.1021/acsmedchemlett.7b00112

The synthesis of potent amide-containing phthalazinone H₁ histamine receptor antagonists is described. Three analogues 3e, 3g, and 9g were equipotent with azelastine and were longer-acting in vitro. Amide 3g had low oral bioavailability, low brain-penetration, high metabolic clearance, and long duration of action in vivo, and it was suitable for once-daily dosing intranasally, with a predicted dose for humans of approximately 0.5 mg per day.

Full text of DOI:10.1021/acsmedchemlett.7b00112

Letter
pubs.acs.org/acsmedchemlett
Design of Phthalazinone Amide Histamine H1 Receptor Antagonists
for Use in Rhinitis
Panayiotis A. Procopiou,*,† Alison J. Ford,§ Paul M. Gore,† Brian E. Looker,† Simon T. Hodgson,†
Duncan S. Holmes,† Sadie Vile,† Kenneth L. Clark,§ Ken A. Saunders,§ Robert J. Slack,§
James E. Rowedder,‡ and Clarissa J. Watts#
†Medicinal Chemistry, §Respiratory Biology, ‡R&D Platform Technology and Science, and #Drug Metabolism and Pharmacokinetcs,
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
S
* Supporting Information
ABSTRACT: The synthesis of potent amide-containing phthalazinone H1 histamine
receptor antagonists is described. Three analogues 3e, 3g, and 9g were equipotent with
azelastine and were longer-acting in vitro. Amide 3g had low oral bioavailability, low
brain-penetration, high metabolic clearance, and long duration of action in vivo, and it
was suitable for once-daily dosing intranasally, with a predicted dose for humans of
approximately 0.5 mg per day.
KEYWORDS: Histamine H1 receptor antagonist, allergic rhinitis, once-daily dosing, topical application, phthalazinone
Chart 1. Representative Oral and Intranasal H1 Receptor
Antagonists Used Clinically, Single-Ligand Histamine H1H3
Dual Receptor Antagonist 1, and Selective H1 Receptor
Antagonists 2a−c Based on a Phthalazinone Scaffold
llergic rhinitis affects globally around 500 million people,
Awith high prevalence in the industrialized world.
Furthermore, it has been steadily increasing with a near
quadrupling of primary care consultations over the last 50
years.1,2 Rhinitis symptoms include nasal itching (pruritus),
irritation and repetitive sneezing, rhinorrhea, nasal congestion,
headache, irritation of the throat, watering of the eyes
(epiphora), and edema. Nasal congestion may become more
pronounced leading occasionally to breathing through the
mouth and snoring3 and loss of smell (hyposmia).4
Small molecule pharmacotherapy for allergic rhinitis includes
antihistamines, corticosteroids, leukotriene antagonists, decon-
gestants, mast cell stabilizers, and anticolinergics.2,3,5 The most
commonly used medicines for the treatment of rhinitis are
antihistamines, which are H1 receptor antagonists (inverse
agonists).6,7 These were introduced several decades ago, and
although they were effective, they caused sleepiness due to their
ability to interact with brain H1 receptors responsible for
wakefulness. Oral second-generation antihistamines possessing
polar carboxylic acid groups (fexofenadine and cetirizine, Chart
1) have reduced brain-penetration and a corresponding
reduction in their side-effects. Nasal congestion is one symptom
that second generation antihistamines are not treating
effectively; therefore, they are frequently used in combination
with α-adrenergic agonist decongestants, for example, pseu-
doephedrine. However, these decongestants have their own
side-effects, such as hypertension. A further advancement in
treating rhinitis was the development of topical treatments,
such as azelastine and olopatadine hydrochlorides. The
advantage of intranasal treatments is the attainment of a higher
concentration of drug directly to the nasal cavity with reduced
systemic side-effects due to the lower dose used. In order to
avoid absorption of the portion of the dose that is swallowed,
topical treatments must exhibit low oral absorption. Azelastine
hydrochloride dosed intranasally is used in allergic rhinitis
patients not responding well to fexofenadine and loratadine,
and is more effective than cetirizine.8,9 Olopatadine hydro-
chloride nasal spray has been approved in the US and has
comparable efficacy to azelastine, same duration of action (12
h), and slower onset of action (30 min vs 15 min), but also
suffers from the same side-effects, dysgeusia (bitter taste),
Received: April 5, 2017
Accepted: April 21, 2017
Published: April 21, 2017
© XXXX American Chemical Society
A
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a
headache, nose-bleeds (epistaxis), and drowsiness (somno-
lence).10
Scheme 1. Synthesis of Amides 3 and 9
We have previously disclosed our work in optimizing
histamine H1 receptor antagonists containing a phthalazinone
scaffold and also the development of single-compound dual H1
and H3 receptor antagonists, leading on to our clinical
candidate 1 (Chart 1).11 We have also published on
phthalazinones 2a−c and their more hydrophilic aza- and
diaza-analogues.12 Rhinitis is closely linked to other allergic
diseases, such as asthma, rhinosinusitis, and conjuctivitis. One
disadvantage of allergic rhinitis treatment using antihistamine
monotherapy is the neglect of other comorbidities. In general,
the most potent anti-inflammatory agents are the glucocorti-
coids, such as fluticasone furoate, which is approved for the
once-daily treatment of allergic rhinitis. In this publication we
describe our efforts in identifying a long-acting, potent human
H1 receptor antagonist, which could either be used in
combination with an H3 receptor antagonist or with fluticasone
furoate.13,14
We have previously reported on the identification of a
channel in the 7TM bundle,11 which was approached from the
2a pyrrolidine nitrogen. We have exploited this finding by
appending the H3 fragment and the butyl linker to construct
the H1H3 dual receptor antagonist 1.11 Highly potent H1
antagonists were obtained with simple straight chain alkyl
substituents on the pyrrolidine nitrogen of 2a, such as 2b and
2c. The ether oxygen of 2c was thought to have formed an H-
bond with a Tyr of TM7.
a
Reagents and conditions: (a) N-(2-bromoethyl)phthalimide, K2CO3,
2-butanone, 80 °C, 18 h, 87%; )b) NH2NH2·H2O, EtOH, 80 °C, 1.25
h, 77%; (c) RCO2H, TBTU, Et3N, DMF, 2 h, or RCOCl, Et3N, DCM;
(d) 2,2,2-trifluoro-N-(2-iodoethyl)acetamide, DIPEA, 2-butanone,
100%; (e) (i) MeI, K2CO3, DMF; (ii) K2CO3, H2O, MeOH, 29%
(two steps).
a
Scheme 2. Synthesis of Amides 10b and 10f
At the outset we were interested in a very potent, homochiral
H1 antagonist suitable for a low-dose, small-volume intranasal
delivery with a 24 h duration of action. We have previously
shown that the (R)-enantiomer of 2a was the higher affinity
enantiomer.11 We envisioned replacing the ether oxygen of 2c
with an amide group, which might provide an even tighter H-
bond leading to increased affinity and/or duration, but also
introduce metabolic instability leading on to increased
clearance, a requirement for topical administration. Further-
more, introducing an electron withdrawing group, such as the
amide functionality, was expected to lower the pKa of the
pyrrolidine nitrogen, thereby increasing selectivity against
hERG.12,15,16
Chemistry. The target amides 3 were prepared by the three-
step synthetic route outlined in Scheme 1. The (R)-enantiomer
of 411 was alkylated with N-(2-bromoethyl)phthalimide to give
5 in 87% yield, deprotected using hydrazine to give amine 6
(77% yield), and then acylated with a variety of carboxylic acids
using TBTU to provide amides 3d,f,g,h or with the
corresponding acid chloride to give 3a,b,c,e. Alkylation of 4
with 2,2,2-trifluoro-N-(2-iodoethyl)acetamide gave the trifluor-
oacetamide 7, which was alkylated with iodomethane and
deprotected to provide the secondary amine 8. The latter was
acylated with 4-methoxybutanoic acid and TBTU to give the
tertiary amide 9g.
a
Reagents and conditions: (a) BrCH2CH2CH2CO2Et, K2CO3, DMF,
150 °C, microwave, 110 min, 51%; (b) 2 M NaOH, H2O, MeOH, 20
°C, 1 h, 91%; (c) nPrNH2 for 10b or MeOCH2CH2NH2 for 10f,
TBTU, Et3N, DMF, 47−68%.
receptor,17 adrenergic α1A and α1B receptor affinity,18 and
hERG activity,18 and the data are summarized in Table 1.
Azelastine, which was used as a standard, exhibited high affinity
for both the H1 receptor (pKi = 8.9) and the hERG channel
(dofetilide binding assay pIC50 = 7.0).11 The straight chain
amides 3a and 3b were equipotent to azelastine; however, they
were less selective for the α1A and α1B receptors and were
rejected. The branched amides 3c and 3d were less potent than
azelastine, but also suffered from reduced selectivity for the α1A
and α1B receptors and were rejected too. The introduction of an
oxygen atom in the unbranched amide chain was used to probe
for additional H-bond interactions. The tetrahydropyran
analogue 3h, like the other branched amides, was less potent
and was rejected. The reverse amide 10b had high hERG
affinity and was also rejected.
In addition to the above assays, another lower-throughput
version of the H1 assay was used, which provided more precise
pA2 values and an indication of the analogues’ duration of
action.11 The data for phthalazinone analogues 3a−h, 9g, 10b
and 10f are summarized in Table 2 and compared to azelastine
(pA2 9.7). Their duration of action in vitro is expressed as
faster, slower, or no-difference wash-out time relative to
azelastine. Slower wash-out equated to longer duration of
action than azelastine, whereas faster wash-out to shorter
duration. Analogues 3f and 10f were rejected based on their
lower affinity than azelastine in the secondary assay (Table 2).
The remaining compounds 3e, 3g, and 9g were equipotent with
azelastine, exhibited a longer duration of action in vitro, and
In addition to amides 3a−h and 9g, alternative amides 10b,h
where the acyl part of the amide group is reversed (closer to the
pyrrolidine basic nitrogen) were prepared and their synthesis is
outlined in Scheme 2. These compounds were synthesized by
alkylation of pyrrolidine 4 with ethyl 4-bromobutyrate to give
ester 11, which was converted to carboxylic acid 12 by base
hydrolysis and finally converted to amides 10b and 10f.
Results and Discussion. The biological screens used to
assay the compounds were reported previously11 and include
H1 receptor affinity using recombinant human histamine H1
B
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a
substrate and that low brain exposure would be maintained.
The trifluoroacetate and hydrochloride salts of 3g were
progressed to the male CD Sprague−Dawley rat and Beagle
dog pharmacokinetic studies in vivo. The compounds were
dosed as solutions in H2O−PEG200−DMSO (50:45:5). Each
compound was dosed iv at 1 mg/kg (both species) and po at 3
mg/kg for the rat and 2 mg/kg for the dog. In addition one dog
was also dosed subcutaneously (Table 4, in SI section).
Following intravenous administration of 3g blood clearance
(Clb) was high compared to liver blood flow (LBF) (means of
66 and 31 mL/min/kg in rat and dog, respectively) and the
steady-state volume of distribution (Vss) was low to moderate
(means of 0.7 and 1.1 L/kg in rat and dog, respectively). The
resultant elimination half-life was short (means of 0.2 and 0.5 h
in rat and dog, respectively). Elimination of parent compound
via urine was low following oral, subcutaneous, and intravenous
administration of 3g·HCl to the dog (nondetectable following
oral administration and <3% following subcutaneous and
intravenous administration). Following oral administration of
3g bioavailability was low (<3% for rat and <8% for dog). Oral
bioavailability in the rat was assessed at 3 mg/kg (negligible
bioavailability) and 300 mg/kg (<3% bioavailability). At the
higher dose in addition to low bioavailability the blood
concentrations were highly variable between rats. Absorption
of 3g into the hepatic portal vein following oral administration
to the rat was also shown to be low (3 to 7% using a 3 mg/kg
dose). Following subcutaneous administration of 3g to the dog
bioavailability appears complete. These data suggest that the
low in vivo bioavailability may be due to high first-pass
metabolism (both intestinal and hepatic) rather than poor
intestinal permeability. The bioavailability of azelastine in the
rat and dog11 was higher than that of 3g, whereas in humans
the reported azelastine bioavailability is 40%.19 The in vitro rate
of metabolism was high for 3g·HCl using liver microsomes
from mouse (50 mL/min/g tissue), rat (50 mL/min/g), dog
(12 mL/min/g), and human (25 mL/min/g). The high in vitro
hepatic clearance data is in agreement with the high in vivo
clearance data for rat and dog. The in vitro rate of metabolism
for 3g·HCl was also significant using intestinal microsomes
from rat (12 mL/min/g), monkey (>50 mL/min/g), and
human (11 mL/min/g). These data are in agreement with the
low bioavailability/absorption observed following oral admin-
istration to the rat, despite 3g showing good cell permeability.
Incubation of 3g·HCl (10 μM initial concentration) with
cryopreserved human hepatocytes for 3 h resulted in extensive
metabolism of the compound, detecting at least 16 metabolites.
Of the metabolites, labeled M1 to M16 according to their
retention time on the LC−MS/MS chromatogram, M1 was the
first compound to elute off the column. The metabolites ranged
from oxidation of the pyrrolidine ring (lactam formation,
dehydrogenation of pyrrolidine ring), amide hydrolysis, N- and
O-dealkylation, and glucuronidation. The structures of these
metabolites are shown in Chart 2 in the SI section together
with their relative abundance. The levels of the two acyl
glucuronide metabolites were low compared to the overall
metabolic profile. No evidence was found for the formation of
N-oxide or glutathione conjugated metabolites. Some second-
generation antihistamines are substrates and modulators of
CYP450, in particular the subtype CYP3A4. The inhibitory
potential of 3g was assessed for the five major human CYP450
enzymes (CYP1A2, >100 μM; CYP2C9, 6.7 μM; CYP2C19, 27
μM; CYP2D6, 1.7 μM; CYP3A4, 1.3 μM). Compound 3g did
not cause time-dependent inhibition with CYP2D6, but a
Table 1. Antagonist Affinity at the Human H1 Receptor
(FLIPR Assay), the α1A and α1B Receptors (in Fibroblast
Cells by Means of Plate-Based Calcium Imaging), Measured
logD at pH 7.4, and hERG Binding Affinity
hERG
pIC50
compd
H1 pKi (n) α1A pKi (n) α1B pKi (n) logD7.4
Azelastine
8.9 0.0
(364)
7.3 0.0
(145)
7.3 0.0
(97)
2.3
3.4
3.7
3.2
3.9
3.1
3.2
3.5
3.3
3.5
3.3
2.5
7.0 (116)
3a·TFA
3b·TFA
3c·TFA
3d·TFA
3e·TFA
9.2 0.2
(4)
7.8 0.4
(2)
7.8 0.1
(2)
6.7 (1)
9.1 0.1
(4)
7.5 0.1
(2)
7.1 0.4
(2)
7.2 0.3
(2)
8.4 0.1
(10)
8.4 0.1
(5)
7.2 0.3
(2)
6.4 0.0
(2)
8.4 0.1
(6)
7.1 0.1
(3)
7.1 (1)
7.3 0.1
(2)
8.9 0.4
(2)
7.3 (1)
7.5 (1)
6.7 (1)
3f·HCO2H 9.1 0.1
8.1 0.1
(2)
7.2 0.1
(2)
6.4 (1)
(4)
3g
8.5 0.0
(4)
7.3 0.1
(9)
7.0 0.2
(8)
6.4 0.0
(4)
3h·TFA
9g·TFA
10b·HCl
10f·HCl
8.0 0.1
(6)
6.7 0.0
(3)
6.1 0.0
(2)
8.5 0.1
(4)
7.5 0.1
(2)
6.9 (1)
6.4 0.1
(4)
8.3 0.1
(5)
8.0 0.3
(2)
7.5 0.2
(3)
8.8 (1)
9.1 0.1
(12)
8.2 0.1
(6)
8.0 0.1
(6)
7.4 0.1
(3)
a
Mean SEM of estimated functional pKi for n < 3; the SEM is the
SD; n = number of experiments
Table 2. Antagonist Affinity at H1 Receptor (pA2) and
Duration of Action in Vitro
a
compd
pA2 SEM
n
wash-out
Azelastine
3a·TFA
3b·TFA
3c·TFA
3d·TFA
3e·TFA
3f·HCO2H
3g
9.7 0.1
9.6 0.2
9.2 0.3
9.88 0.15
9.5 0.1
9.7 0.1
9.3 0.1
9.74 0.09
9.1 0.1
10.0 0.3
9.8 0.2
9.3 0.1
19
6
reference
no difference
slower
2
8
slower
13
14
10
20
13
12
9
slower
slower
slower
slower
3h·TFA
9g·TFA
10b·HCl
10f·HCl
slower
slower
slower
8
slower
a
All pA2 values calculated from curve shifts generated after 30 min
incubation time at 100 nM antagonist concentration.
were therefore of interest for further examination. Analogue 3g
was historically made before the other two compounds and
hence was investigated first.
Pharmacokinetics Amide 3g. TFA had low in vivo CNS
penetration in the rat (brain−blood ratio of 0.18 and mean
brain concentration of 121 ng/g, n = 3) assessed in samples
taken 5 min after an intravenous bolus dose of 1 mg/kg. High
in vitro plasma protein binding was observed in four species
(rat, guinea pig, dog, and human), and blood cell association of
3g·HCl was low to moderate in the same species (Table 3, in SI
section). The membrane permeability of 3g·HCl across MDCK
cells in the presence and absence of a P-glycoprotein (PgP)
inhibitor was 220 nm/s at pH 7.4 when incubated at a
concentration of 2.5 μg/mL, indicating that 3g is not a PgP
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significant change in IC50 with time was observed using
CYP3A4 (mean fold change of 5). In the Cypex cDNA screen
Azelastine also shows time-dependent inhibition of CYP3A4.
Effect of 3g on Histamine-Induced Nasal Congestion in
Vivo. The standard Buxco whole body plethysmography
technique was used to investigate the effect of intranasally
dosed 3g on histamine-induced nasal congestion in conscious,
unrestrained guinea pigs, which were previously sensitized with
ovalbumin and aluminum hydroxide intranasally over a 3-week
period prior to the study. Recording PenH (enhanced pause)
AUC over 40 min following bilateral histamine challenge (15 or
10 mM, 25 μL/nostril, under light isoflurane anesthesia) allows
for the assessment of efficacy and duration of action of
intranasally dosed antihistamines.20
Compound 3g showed significant inhibition of histamine-
induced congestion, compared to a vehicle-pretreated/hista-
mine challenged control group, at 1 and 24 h after
administration of a 1 mg/mL solution (25 μL/nostril). In
contrast, azelastine did not demonstrate a similar duration of
action at the same concentration (Figure 1). Further studies
very low. The concentration of 3g was less than the limits of
quantification for the assays used in all assayed blood samples
taken from guinea pigs showing a pharmacological response in
a histamine induced nasal congestion model following
intranasal administration of 3g. In order to appreciate
toxicology risks/overage the pharmacokinetic profiles in
preclinical species following intravenous administration were
used to give estimates of the maximum systemic exposure in
humans if most of the intranasal dose was swallowed and
bioavailability was complete. For the predictions a standard
absorption rate was assumed; the volume of distribution was
kept constant and the elimination rate was scaled based on liver
blood flow. Based on these simulations the maximum estimates
for exposure in humans following a 0.5 mg dose are a total drug
Cmax of 1.4 and 1.6 ng/mL (based on rat and dog data,
respectively) and an AUC0−24 of 4.1 and 4.8 h.ng/mL (based
on rat and dog data, respectively).
Specificity Profiling of 3g. The selectivity of 3g for the
human H1 receptor over the other three histamine receptors
(H2, H3, and H4) was evaluated in binding assays on
recombinantly expressed histamine receptors, using measure-
ments of inhibition of histamine stimulated cAMP in HEK-293
cell membranes (for H2), inhibition of histamine stimulated
GTPγS binding in CHO cell membranes (for H3), and
inhibition of radiolabeled histamine binding in HEK-293 cell
membranes (for H4) and found to show >1000-fold specificity.
Compound 3g had a pA2 for H1 = 9.4 (n = 54), pKi for H2 5.1
(n = 2), pKi for H3 < 6.2 (n = 29), and pKi for H4 < 6.4 (n = 2).
The selectivity of 3g and azelastine against 50 different
receptors, ion channels, enzymes, and transporters was
evaluated in the CEREP Specificity Screen in agonist or
antagonist mode as appropriate. Compound 3g (at 1 μM)
caused >50% inhibition at 6 targets in this screen (α1, D1, D2S,
5-HT2A, 5-HT6, and 5-HT7), indicative of some off-target
activity; a similar profile was seen with azelastine, which caused
>50% inhibition at 10 targets, four of which overlapped with 3g.
Further work was performed to quantify and compare the
specificity profile of 3g relative to H1 receptor (pA2 9.4), which
showed >100-fold specificity, apart from the human 5-HT2B
receptor (pKi 7.8 in antagonist mode; 40-fold selectivity for
H1). This is not considered to be an issue since azelastine,
which is used clinically, had the same pKi value in the same
assay. In addition 3g has lower bioavailability than azelastine
suggesting that systemic exposure after intranasal adminis-
tration is likely to be negligible. Compound 3g also showed
significant affinity (pIC50 = 6.4) in the hERG binding assay,
however, this was lower than azelastine (pIC50 = 7.0).
Furthermore, 3g was clean in the rabbit ventricular wedge
assay (up to 10 μM), suggesting that the risk of 3g-induced QT
prolongation is low at the predicted exposure levels.
Figure 1. Duration of action of azelastine and 3g in the guinea pig
model of nasal congestion. Animals were exposed to histamine at 1
and 24 h after an intranasal dose of 1 mg/mL of 3g·TFA, 3g·HCl, or
azelastine. Mean SEM (n = 16−27 per group). *p < 0.05 compared
to histamine control group; #p < 0.05 compared to vehicle/PBS
control group. Bar indicates p < 0.05 individual comparison as
indicated. ANOVA with posthoc Hochberg analysis.
indicated that duration of action is less than 48 h following
administration of a 1 mg/mL solution of 3g (data not shown).
Neither 3g nor azelastine produced a procongestant response at
any time point studied (effect on baseline PenH, data not
shown). Arterial blood samples taken immediately following
evaluation of nasal congestion (4 or 25 h after intranasal
dosing) do not show any systemic exposure from 1 mg/mL of
3g in these studies, which indicates a low bioavailability by this
route and a local mechanism of action.
The mutagenic and clastogenic potential of 3g was assessed
in the AMES test, Mouse Lymphoma screen and DEREK
structure−activity relationship in silico analysis. No evidence of
mutagenic potential was detected in the mini-AMES assay up to
the maximum concentrations tested (which were limited by
precipitation and/or toxicity as appropriate in each strain) in
the presence or absence of an in vitro metabolic activation
system (S9-mix). No evidence of clastogenic potential was
detected in the mouse lymphoma screen up to maximum
concentrations of 80 μg/mL for 3 h in the presence of S9-mix
and 40 μg/mL for 24 h in the absence of S9-mix (both
maximum concentrations were limited by cytotoxicity). The
analysis of the molecular structure of 3g using DEREK in silico
In order to determine the cross species activity between
human and guinea pig,20 the effect of 3g on the contractile
response to histamine in guinea pig isolated trachea (i.e., upper
respiratory tract and closest available tissue assay to the nasal
cavity for investigation) and human bronchus was determined
(guinea pig pA2 8.7 and human bronchus 8.2) demonstrating
that the potency was comparable between species.
Predicted Human Dose. The clinical dose of 3g is expected
to be low (approximately 0.5 mg per day) based on the clinical
dose used for azelastine and a comparison of efficacy data from
animal models for 3g and azelastine. Based on high blood
clearance, low oral rat and dog bioavailability, and a low clinical
dose, the systemic exposure to 3g in humans is expected to be
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(2) Greiner, A. N.; Hellings, P. W.; Rotiroti, G.; Scadding, G. K.
Allergic rhinitis. Lancet 2011, 378, 2112−2122.
analysis showed no evidence of overt mutagenic or toxic
potential.
(3) Mandhane, S. N.; Shah, J. H.; Thennati, R. Allergic rhinitis: An
update on disease, present treatments and future prospects. Int.
Immunopharmacol. 2011, 11, 1646−1662.
Conclusion. A series of pyrrolidine amide human H1
histamine receptor antagonists based on the phthalazinone
scaffold were synthesized. Amides 3e, 3g, and 9g were
equipotent with azelastine, the clinical gold-standard (pA2 9.9,
9.7, and 10.0, respectively, vs 9.7 for azelastine), and had a
longer duration of action than azelastine in vitro. Amide 3g was
selective for the H1 receptor with >1000-fold selectivity over
the other three histamine receptors. Furthermore, 3g had
significantly longer duration of action in vivo than azelastine in
a nasal congestion model. The bioavailability of 3g was low,
which minimizes the potential for side-effects from the
swallowed fraction of the intranasal dose. Mean brain
concentration following iv dosing in the rat was low, which
coupled with the low bioavailability, should limit the potential
for any CNS related side-effects. The in vitro rate of
metabolism for 3g was high using human liver and intestinal
microsomes, and in hepatocytes. A number of metabolites in
human hepatocytes were detected by LC−MS/MS and
includes metabolites from oxidation of the pyrrolidine ring
(lactam formation), amide hydrolysis, N- and O-dealkylation,
and glucuronidation. High in vivo clearance was observed in the
rat and dog. Amide 3g had a lower affinity than azelastine for
the hERG channel, and high human plasma-protein binding.
CYP3A4 was inhibited by 3g in a time-dependent manner;
however, azelastine behaves similarly in the Cypex cDNA
screen. As the bioavailability of 3g is low, coadministration with
other drugs is unlikely to cause any significant drug−drug
interactions in the clinic. In summary, 3g is suitable for
progression as an intranasal candidate for the treatment for
allergic rhinitis, either as a monotherapy or in combination with
an anti-inflammatory steroid, such as fluticasone furoate.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00112.
■
S
(16) Alberati, D.; Hainzl, D.; Jolidon, S.; Krafft, E. A.; Kurt, A.; Maier,
A.; Pinard, E.; Thomas, A. W.; Zimmerli, D. Discovery of 4-
substituted-8-(2-hydroxy-2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]-
decan-1-one as a novel class of highly selective Gly T1 inhibitors with
improved metabolic stability. Bioorg. Med. Chem. Lett. 2006, 16, 4311−
4315.
Experimental details on all compounds and characteriz-
ing data, Tables 3 and 4, and Chart 2 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: pan.a.procopiou@gsk.com.
ORCID
Panayiotis A. Procopiou: 0000-0001-5907-116X
Notes
The authors declare no competing financial interest.
■
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ACKNOWLEDGMENTS
■
We thank Daniel Parton and Alasdair J. Carey for technical
assistance, the late Michael Kranz for the modeling work, Dr.
Bill Leavens for the HRMS measurements, Anne Jones for the
metabolite identification work, and Dr. Christopher Browning
and Ms. Linda J. Russell for the in vitro duration of action
studies.
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DOI: 10.1021/acsmedchemlett.7b00112
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX